Treatment of amd using aav sflt-1

ABSTRACT

The present disclosure provides compositions and methods for the prevention or treatment of ocular neovascularization, such as AMD, in a human subject, by administering subretinally a pharmaceutical composition comprising a pharmaceutically effective amount of a vector comprising a nucleic acid encoding soluble Fms-related tyrosine kinase-1 (sFlt-1) protein to the human subject.

This application is a Continuation application of U.S. patentapplication Ser. No. 13/889,275 filed on May 7, 2013; which applicationclaims priority under 35 USC §119(e) to U.S. Provisional Application No.61/647,461, filed May 15, 2012, U.S. Provisional Application No.61/670,535, filed Jul. 11, 2012, U.S. Provisional Application No.61/678,555, filed Aug. 1, 2012, U.S. Provisional Application No.61/775,440, filed Mar. 8, 2013, each of which are incorporated byreference in their entirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted inASCII format via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jun. 14, 2013, is named43016-702.201_SL.txt and is 86,152 bytes in size.

BACKGROUND OF THE DISCLOSURE

Age-related macular degeneration (AMD) is one of the leading causes ofvision irreversible damage in people over the age of 50 years. AMD isclinically divided into two types as “dry” and “wet”. The wet form ofAMD may develop rapidly and often results in blindness. The pathologicalchanges of the disease may cause severe visual impairment. Themanifestations of AMD may include, but is not limited to retinal pigmentepithelial cells (RPE) dysfunction and choroidal neovascularization(CNV) in the macular area. Fluid leakage, RPE or neural epithelialdetachment and bleeding from ruptured blood vessels can occur in severecases. It has been found that many cellular factors play important rolesin regulation in CNV generation, among which may include but are notlimited to vascular endothelial growth factor (VEGF), VEGF receptor(VEGFR), platelet-derived growth factor (PDGF), hypoxia inducible factor(HIF), angiopoietin (Ang) and other cytokines, mitogen-activated proteinkinases (MAPK) and others.

One currently approved treatment for wet AMD is Lucentis®. Lucentis® isan anti-angiogenesis agent and targets all isoforms of VascularEndothelial Growth Factor (VEGF). Clinical studies have shown improvedor stable vision in approximately 95% of patients administeredLucentis®, compared to approximately 60% of the patients who receivedsham treatment. Although Lucentis® is the first approved agent toimprove vision it requires intravitreal administrations every 4 weeksfor optimal visual benefit. Eylea® is another VEGF inhibitor that hasbeen approved to treat wet AMD. Eylea® also requires frequentintravitreal injections every 4-8 weeks for optimal visual benefit.Intravitreal routes of administration may increase risks for seriouscomplications such as infectious endophthalmitis and retinal detachment,for which cumulative risk increases with repeated administrations.Increased intraocular pressure, traumatic cataract, and retinal tearshave also been reported. Finally, with a treatment that is delivered byan ophthalmologist, treatment frequency determines the burden to thepatient, physician, and health system in general and to the extentpossible should be reduced. The limitations of currently availabletherapy for CNV secondary to AMD have created a need in the art foralternative approaches which address the high frequency of treatmentsrequired and the invasiveness of the treatment procedure.Neovascularization involving VEGF elevation can also lead to otherocular pathologies, such as diabetic retinopathy, diabetic macular edema(DME), and retinal vein occlusions (RVO). These diseases lead to retinalneovascularization and vision loss. VEGF inhibitors such as Lucentis®have demonstrated efficacy in DME and RVO, and, like with wet AMD,require frequent intravitreal administration in order to maintainbenefit.

SUMMARY OF THE DISCLOSURE

The present disclosure provides compositions and methods for treatingCNV, such as found in the wet form of AMD, in a human subject.

In one aspect, the present disclosure provides compositions and methodsfor treating AMD in a human subject, comprising: administeringsubretinally a pharmaceutical composition comprising a pharmaceuticallyeffective amount of a VEGF inhibitor to a human subject in need oftreatment for AMD. In one aspect, the pharmaceutical compositioncomprises a recombinant virus. In another aspect, the VEGF inhibitorcomprises a nucleic acid encoding soluble Fms-related tyrosine kinase-1(sFLT-1) protein.

In one aspect, the present disclosure provides compositions and methodsfor the prevention of CNV in human subjects with AMD, comprising:administering subretinally a pharmaceutical composition comprising apharmaceutically effective amount of a recombinant virus comprising anucleic acid encoding soluble Fms-related tyrosine kinase-1 (sFLT-1)protein to a human subject in need of a treatment for AMD.

In some aspects, the virus is selected from adeno-associated virus(AAV), helper-dependent adenovirus, retrovirus, herpes simplex virus,lentivirus, poxvirus, hemagglutinatin virus of Japan-liposome (HVJ)complex, Moloney murine leukemia virus, and HIV-based virus. In someaspects, the AAV capsid or inverted terminal repeats (ITRs) is selectedfrom the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAV10, AAV11, AAV12, rh10, and hybrids thereof.

In some aspects, the recombinant virus comprises a promoter selectedfrom cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter,MMT promoter, EF-1 alpha promoter, UB6 promoter, chicken beta-actinpromoter, CAG promoter, RPE65 promoter and opsin promoter.

In some aspects, the recombinant virus comprises an enhancer.

In some aspects, the recombinant virus comprises an intron or chimericintron.

In some aspects, the recombinant virus comprises a SV40 poly A sequence.

In some aspects, the recombinant virus comprises a human sFlt-1 proteinor a functional fragment thereof.

In some aspects, the recombinant virus is generated from a plasmidcomprising either an ampicillin resistance marker or a non-ampicillinresistance marker.

In some aspects, the recombinant virus comprises bacterial regulatorysequences such as a T7 RNA polymerase promoter.

In some aspects, the recombinant virus lacks bacterial regulatorysequences such as a T7 RNA polymerase promoter.

In some aspects, the recombinant virus comprises a regulatory nucleicacid fragment that is capable of directing selective expression of thesFlt-1 protein or a functional fragment thereof in an eye cell.

In some aspects, the pharmaceutical composition comprises about 1×10⁶ toabout 1×10¹⁵ recombinant viral vector genomes, about 1×10⁷ to about1×10¹⁴ recombinant viral vector genomes, about 1×10⁹ to about 1×10¹²recombinant viral vector genomes, about 1×10¹⁰ to about 3×10¹²recombinant viral vector genomes, or about 1×10¹⁰ to about 3×10¹²recombinant viral vector genomes.

In some aspects, the pharmaceutical composition is administered viasubretinal injection.

In some aspects, the method further comprises administering to the humansubject a pharmaceutically effective amount of a VEGF inhibitor. In someaspects, the VEGF inhibitor comprises an antibody against VEGF or afunctional fragment thereof. In some aspects, the VEGF inhibitorcomprises ranibizumab. In some aspects, the pharmaceutical compositionis administered at least 5, 6, 7, or 8 days after the administering theVEGF inhibitor. In some aspects, the pharmaceutical composition isadministered within 30, 60, or 90 days of administering the VEGFinhibitor.

In some aspects, the VEGF inhibitor is administered for 1 time prior toadministering the pharmaceutical composition comprising the recombinantvirus and 1 to 2 times following administration. In some aspects, theVEGF inhibitor is administered for at least 2 times prior toadministering the pharmaceutical composition and 1 to 2 times followingadministration. In some aspects, the VEGF inhibitor is administered overa period of 6 to 7 weeks.

In some aspects the VEGF inhibitor is an anti-VEGF antibody, such asbevacizumab or ranibizumab. In other aspects the VEGF inhibitor is asoluble receptor, fusion protein, or fragment thereof, such asaflibercept or sFLT01.

In some aspects, the AMD is wet AMD.

In some aspects, AMD is dry AMD.

In some aspects, the human subject is at risk for wet AMD.

In some aspects, the human subject presents symptoms of early stage wetAMD.

In some aspects, at least 3, 5, 10, 15, or 20 treatments of a differentVEGF inhibitor for the treatment of AMD have been previouslyadministered to said human subject

In some aspects, best corrected visual acuity (BCVA) did not improveafter said treatment with ranibizumab.

In some aspects, best corrected visual acuity (BCVA), as measured byETDRS (Early Treatment Diabetic Retinopathy Study) letters, improves bymore than 1 line after said treatment with ranibizumab.

In some aspects, human subject presents symptoms of early stage dry AMD.

In some aspects, treatment is administered at a frequency of at leastbiannually.

In some aspects, administering step is carried out in said human subjectwhere the subject is age 20, 40, 50, 55, or 65 years or older.

In some aspects, administration is to a site outside the fovea.

In some aspects, administration is to one or more cells of thesubretinal space of the central retina.

In some aspects, administration is to one or more cells of the outermacula.

In some aspects, administration is to one or more cells of the innermacula.

In some aspects, administration is to retinal pigment epithelial cells.

In some aspects, administration does not adversely affect centralretinal function or central retinal structure.

In some aspects, administration does not increase systemic levels ofVEGF inhibitor in the human subject.

In some aspects, administration does not increase systemic levels ofsFlt-1 in the human subject.

In some aspects, administering step is carried out simultaneously, orsequentially in both eyes

In some aspects, administering step is carried out in one eye.

In some aspects, administering step is carried out in one eye whenfellow eye presents symptoms of AMD.

In some aspects, administering step is carried out in a human subjectresistant to penicillin.

In some aspects, administering step is carried out in a human subjectsensitive to penicillin.

In some aspects, administering step is carried out in a human subjectallergic to penicillin.

In some aspects, administering step is carried out in a human subjectnot allergic to penicillin.

In some aspects, administering step causes no inflammation of thevitreous is observed by biomicroscopy (BE) and indirect opthalmoscopy(JOE) following the administering step.

In some aspects, administering step does not cause a cytotoxic T cell.

In some aspects, administering step does not cause a cytotoxic T cellresponse a measure by in increase in cytotoxic T cells of less than 10%greater than the baseline range.

In some aspects, T cells do not display an activated effector phenotypefollowing the administering step.

In some aspects, best corrected visual acuity (BCVA) improves by 1, 2,3, 4 or 5 lines or more. as measured by ETDRS (Early Treatment DiabeticRetinopathy Study) letters, following the administering step.

In some aspects, reduction in neovascularization is observed usingFluorscein Angiography (FA) following the administering step

In some aspects, frequency of administration of ranibizumab is reducedto less than 12 doses per year. In some aspects, frequency ofadministration of aflibercept is reduced to less than 6 doses per year.

In some aspects, ranibizumab or aflibercept or other VEGF inhibitor isadministered with reduced frequency or no longer administered.

In some aspects, the virus comprises a sFLT-1 gene or a functionalfragment thereof with ≧90% sequence homology to the human sFLT-1 genesequence.

In some aspects, the virus administered comprises a sFLT-1 gene, genevariant or gene fragment.

In some aspects, no vector is detected in the human subject's tear,blood, saliva or urine samples 7, 14, 21 or 30 days after administeringthe pharmaceutical composition.

In some aspects, the presence of the viral vector is detected by VCR orELISA.

In some aspects, the sFLT-1 protein levels in the vitreous of the humansubject is about 500-5,000 pg/ml, about 600-4,000 pg/ml, about 800-3,000pg/ml about 900-2,000 pg/ml, or about 1,000-1,800 pg/ml 7, 14, 21 or 30days after administering the pharmaceutical composition. In someaspects, the sFlt-1 protein level, which may also be called the sFlt-1protein concentration, in the vitreous of the human subject is elevatedat 7, 14, 31, 30, 60, 90, 180, 270 and 365 days after administering thepharmaceutical composition.

In some aspects, the human subject shows no clinically significantretinal toxicity as assessed by serial ophthalmic examinations overleast a two months period.

In some aspects, no superficial, anterior segment or vitreousinflammatory signs are present in the human subject over least a twomonths period.

In some aspects, the human subject does not require rescue treatmentwith a VEGF inhibitor at least 120 days post administering therecombinant viruses. In some aspects, the human subject does not requirerescue treatment with a VEGF inhibitor at least 180 days or at least 210days post administering the recombinant viruses. In some aspects, thehuman subject does not require rescue treatment with a VEGF inhibitorfor at least 270 days after administering the recombinant viruses. Insome aspects, the human subject does not require rescue treatment with aVEGF inhibitor for at least 365 days after administering the recombinantviruses.

In some aspects, there is no evidence of visual acuity loss, IOPelevation, retinal detachment, or any intraocular or systemic immuneresponse in said human subject at least 180 days or at least 210 dayspost said administering the recombinant viruses. In some aspects, thereis no evidence of visual acuity loss, IOP elevation, retinal detachment,or any intraocular or systemic immune response in said human subject atleast 365 days after administering the recombinant viruses.

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising about 1×10⁶ to about 1×10¹⁵ recombinant viruses,wherein each of the recombinant virus comprises a nucleic acid encodingsoluble Fms-related tyrosine kinase-1 (sFlt-1) protein.

In some aspects, the disclosure provides for a method for the treatmentor prophylaxis of ocular neovascularization in a human subjectcomprising: administering to one or more subretinal sites apharmaceutically effective amount of a pharmaceutical compositioncomprising a nucleic acid encoding sFLT-1 to a human subject in need oftreatment.

In some aspects, the disclosure provides for a human subject that has oris suspected of having one or more conditions selected from the groupconsisting of: age-related macular degeneration (AMD), wet-AMD, dry-AMD,retinal neovascularization, choroidal neovascularization and diabeticretinopathy. In some cases the human subject has or is suspected ofhaving one or more conditions selected from the group consisting of:proliferative diabetic retinopathy, retinal vein occlusion, centralretinal vein occlusion, branched retinal vein occlusion, diabeticmacular edema, diabetic retinal ischemia, ischemic retinopathy anddiabetic retinal edema.

In some aspects, the disclosure provides for a pharmaceuticalcomposition comprising a recombinant virus, the virus selected from thegroup consisting of: adeno-associated virus (AAV), adenovirus,helper-dependent adenovirus, retrovirus, herpes simplex virus,lentivirus, poxvirus, hemagglutinatin virus of Japan-liposome (HVJ)complex, Moloney murine leukemia virus, and HIV-based virus.

In some aspects, the disclosure provides for a nucleic acid encoding thesFLT-1 which is operatively linked to a promoter selected from the groupconsisting of: cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV)promoter, MMT promoter, EF-1 alpha promoter, UB6 promoter, chickenbeta-actin promoter, CAG promoter, RPE65 promoter and opsin promoter.

In some aspects, the disclosure provides sFLT-1 nucleic acid, whereinthe sFLT-1 encodes at least 1 dimerization domain. In some cases thesFLT-1 nucleic acid does not contain a prokaryotic regulatory sequence.In some cases the sFLT-1 nucleic acid does contain a prokaryoticregulatory sequence.

In some aspects, the disclosure provides for a pharmaceuticalcomposition comprising a virus or a plasmid.

In some aspects, the disclosure provides for administration of one ormore treatments of a VEGF inhibitor to the human subject. In some casesthe VEGF inhibitor is administered within 30, 90, or 180 days ofadministration of the pharmaceutical composition. In some cases thepharmaceutical composition of the disclosure and VEGF inhibitor areadministered at least 24 hours apart.

In some aspects, the disclosure provides for a pharmaceuticalcomposition administered to a human subject at least 55 years old.

In some aspects, the disclosure provides for administering thepharmaceutical composition outside the fovea.

In some aspects, the disclosure provides for the best corrected visualacuity (BCVA) of the human subject, to improve by at least 1, 2, 3, 4 or5 lines as measured by ETDRS (Early Treatment Diabetic RetinopathyStudy) letters following the administering of the pharmaceuticalcomposition.

In some aspects, the disclosure provides for the best corrected visualacuity (BCVA) to decrease by fewer than 15 letters as measured by ETDRS(Early Treatment Diabetic Retinopathy Study) following the administeringof the pharmaceutical composition.

In some aspects, the disclosure provides for administering thepharmaceutical composition under conditions selected from the groupconsisting of: administering the pharmaceutical composition in one eye,administering the pharmaceutical composition sequentially in two eyes,and administering the pharmaceutical composition simultaneously in twoeyes.

In some aspects, the disclosure provides for a reduction inneovascularization as observed by a Fluorscein Angiography (FA) followsthe administering of the pharmaceutical composition.

In some aspects, the disclosure provides for no superficial, anteriorsegment or vitreous inflammatory signs are present in the human subjectat least 1 week after injection.

In some aspects, the disclosure provides for no superficial, anteriorsegment or vitreous inflammatory signs are present in the human subjectat 1 week or at 3, 6, 9 or 12 months after administration of thepharmaceutical composition.

In some aspects, the disclosure provides for the human subject not torequire rescue treatment for at least 30, 60, 90, 120, 180, 270 or 365days after the administering of the pharmaceutical composition.

In some aspects, the disclosure provides for the human subject toexperience no visual acuity loss, IOP elevation, retinal detachment,intraocular or systemic immune response after administering thepharmaceutical composition.

In some aspects, the disclosure provides for no increased anti-AAVcytotoxic T cell response is measured following the administering step.

In some aspects, the disclosure provides for no virus detected in thehuman subject's blood, saliva or urine samples, 3, 7, 14, 21 or 30 daysafter administering the pharmaceutical composition.

In some aspects, the disclosure provides for sFT-1 protein levels in thevitreous of the human subject to be about 500-5,000 pg/ml, 7, 14, 21,30, 60, 90, 120, 150, 180, 270 or 365 days after administering thepharmaceutical composition in the human subject.

In some aspects, the disclosure provides for the human subject toreceive one or more treatments with VEGF inhibitors prior to theadministering of the pharmaceutical composition.

In some aspects, the disclosure provides for the human subject asresistant to treatment with VEGF inhibitors.

In some aspects, the disclosure provides for a human subject who has notpreviously received a VEGF inhibitor before administering thepharmaceutical composition.

In some aspects, the disclosure provides for administering of thepharmaceutical composition at a frequency less than 3 times a year inthe human subject.

In some aspects, the disclosure provides for administering of thepharmaceutical composition to reduce the frequency of administration ofadditional VEGF inhibitor treatments in the human subject.

In some aspects, the disclosure provides for the concentration of sFLT-1protein in the vitreous of the human subject to be elevated whenmeasured at 7, 14, 21, 30, 60, 90, 120, 150, 180, 270 or 365 days afteradministering of the pharmaceutical composition.

In some aspects, the disclosure provides for a human subject who has thevitreous gel removed prior to or within one day or one week of theadministration of the pharmaceutical composition.

In some aspects, the disclosure provides for a pharmaceuticalcomposition administered using a vitrectomy system that is smaller than20 gauge.

In some aspects, the disclosure provides for a pharmaceuticalcomposition administered using a vitrectomy system that does not requiresutures.

In some aspects, the disclosure provides for a pharmaceuticalcomposition administered using a cannula tip that is smaller than 39gauge.

In some aspects, the disclosure provides for a pharmaceuticalcomposition followed by gas/fluid exchange in the vitreous chamber.

In some aspects, the disclosure provides for the central retinalthickness of the subject not to increase by more than 50 microns, 100microns, or 250 microns within 12 months following treatment with saidpharmacological agent.

In some aspects, the disclosure provides for geographic atrophy not toprogress in the diseased eye of the human subject as compared to thediseased eyes of untreated human subjects.

In some aspects, the disclosure provides for a pharmaceuticalcomposition comprising recombinant viruses or plasmids comprising anucleic acid comprising at least 1 promoter sequence operatively linkedto a sFLT-1 transgene sequence. In some cases the pharmaceuticalcomposition of the disclosure comprises a promoter sequence and thesFLT-1 transgene sequence separated by a sequence greater than 300 basepairs. In some cases the pharmaceutical composition of the disclosurecomprises a promoter sequence and the sFLT-1 transgene sequenceseparated by a UTR sequence. In some cases the UTR sequence comprises atleast 10 base pairs. In some cases, the pharmaceutical compositioncomprises at least 3 linker sequences each comprising at least 50 basepairs.

In some aspects, the disclosure provides for a pharmaceuticalcomposition, wherein the sFLT-1 nucleic acid encodes at least 1dimerization domain.

In some aspects, the disclosure provides for a pharmaceuticalcomposition comprising a promoter sequence selected from the groupconsisting of SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No.20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ IDNo. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 34,SEQ ID No. 35, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, SEQ ID No.39, SEQ ID No. 340, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43, SEQ IDNo. 44, SEQ ID No. 45, SEQ ID No. 46, and SEQ ID No. 47; a sequenceencoding a VEGF inhibitor selected from the group consisting of SEQ IDNo. 102, SEQ ID No. 103, SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106,SEQ ID No. 107 and SEQ ID No. 108; an intron sequence consisting of SEQID No. 48, SEQ ID No. 115, SEQ ID No. 116, SEQ ID No. 117, SEQ ID No.118, and SEQ ID No. 119; a UTR sequence selected from the groupconsisting of SEQ ID No. 91, SEQ ID No. 2, SEQ ID No. 92, SEQ ID No. 93,SEQ ID No. 94, SEQ ID No. 95, SEQ ID No. 96, SEQ ID No. 97, SEQ ID No.98, SEQ ID No. 99, SEQ ID No.100, and SEQ ID No. 101; and a terminationsequence selected from the group consisting of SEQ ID No. 49, SEQ ID No.50, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, and SEQID No. 55.

In some aspects, the disclosure provides for a unit dose of apharmaceutical composition comprising recombinant viruses of 1×10⁶ to1×10¹⁵ vector genomes, wherein the recombinant viruses comprise anucleic acid encoding sFLT-1 operatively linked to a promoter. In somecases the unit dose of the pharmaceutical composition comprises 1×10¹⁰to 3×10¹² vector genomes.

In some aspects, the disclosure provides for a method of generating arecombinant virus in a cell, the method comprising: introducing into acell, a nucleic acid comprising at least 1 promoter sequence operativelylinked to an sFLT-1 transgene sequence, an ITR sequence, and UTRsequence; and purifying the recombinant virus. In some cases the UTRsequence is a human UTR sequence. In some cases, the nucleic acidsequence does not contain a beta-lactam antibiotic resistance sequence.In some cases the recombinant virus produces sFLT-1 protein in the rangeof 100-10,000 pg/mL when measured at 72 hours following transduction ofHEK293 cells at a multiplicity of infection (MOI) of 1×10⁶. In somecases, the recombinant virus inhibits proliferation of human umbilicalvascular endothelial (HUVEC) cells.

In some aspects, the disclosure provides for a cell for generatingrecombinant viral vector, the cell comprising at least 1 promoterpolynucleotide sequence operatively linked to a sFLT-1 transgenesequence, an ITR polynucleotide sequence, and a UTR polynucleotidesequence.

In some aspects, the disclosure provides for a nucleic acid comprising asequence encoding sFLT-1 for use in treatment or prophylaxis of ocularneovascularization in a human; wherein said use comprises administeringdirectly to a human subject in need thereof, to one or more sub retinalsites in said human subject, an effective amount of a pharmaceuticalcomposition; wherein said pharmaceutical composition comprises saidnucleic acid.

In some aspects, the disclosure provides the nucleic acid for use,wherein said sFLT-1 is an inhibitor of VEGF and wherein said treating orreducing the likelihood of ocular neovascularization occurs as a resultof VEGF inhibition.

In some aspects, the disclosure provides for the nucleic acid for use,wherein the pharmaceutical composition is capable of elevating levels ofsFLT-1 protein in the vitreous of the human subject after at least 72hours after administration of said pharmaceutical composition to saidhuman subject, compared to levels of sFLT-1 protein in the vitreous ofsaid human prior to said administration.

In some aspects, the disclosure provides for the nucleic acid for use,wherein the nucleic acid comprising said sFLT-1 comprises a recombinantvirus, the virus selected from the group consisting of: adeno-associatedvirus (AAV), adenovirus, helper-dependent adenovirus, retrovirus, herpessimplex virus, lentivirus, poxvirus, hemagglutinatin virus ofJapan-liposome (HVJ) complex, Moloney murine leukemia virus, andHIV-based virus.

In some aspects, the disclosure provides for the nucleic acid for use,wherein the nucleic acid encoding the sFLT-1 is operatively linked to apromoter selected from the group consisting of: cytomegalovirus (CMV)promoter, Rous sarcoma virus (RSV) promoter, MMT promoter, EF-1 alphapromoter, UB6 promoter, chicken beta-actin promoter, CAG promoter, RPE65promoter and opsin promoter.

In some aspects, the disclosure provides for the nucleic acid for use,wherein the nucleic acid is packaged by a virus or is plasmid DNA.

In some aspects, the disclosure provides for the nucleic acid for use,said use further comprising administration of one or more additionalVEGF inhibitors to the human subject in need of treatment or reduction,optionally wherein said additional VEGF inhibitor is ranibizumab orbevacizumab.

In some aspects, the disclosure provides for the nucleic acid for use,said use comprising administering said pharmaceutical composition to ahuman subject at least 50, 55, or 65 years old.

In some aspects, the disclosure provides for the nucleic acid for use,said use comprising administering said pharmaceutical compositionoutside the fovea.

In some aspects, the disclosure provides for the nucleic acid for use,wherein the best corrected visual acuity (BCVA) of the human subject inneed of treatment, improves by at least 1, 2, 3, 4 or 5 lines asmeasured by ETDRS (Early Treatment Diabetic Retinopathy Study) lettersfollowing the administering of an effective amount of the pharmaceuticalcomposition.

In some aspects, the disclosure provides for the nucleic acid for use,wherein the administering of the pharmaceutical composition is performedat a frequency at least once per 3, 6, 9, 12, 18, or 24 months in ahuman subject in need of treatment.

In some aspects, the disclosure provides for the nucleic acid for use,wherein the administering of the pharmaceutical composition is performedat a frequency less than 3 times a year in the human subject or isperformed at a frequency reducing the frequency of administration ofadditional VEGF inhibitor treatments in the human subject.

In some aspects, the disclosure provides for a unit dose ofpharmaceutical composition comprising about 1×10⁶ to 1×10¹⁵ or 1×10¹⁰ to3×10¹² vector genomes. In some aspects, the recombinant viruses comprisea nucleic acid encoding sFLT-1, or a functional fragment thereof,operatively linked to a promoter.

In some aspects, the disclosure provides for a method for the treatmentor prophylaxis of ocular neovascularization in a human subjectcomprising: administering to one or more subretinal sites apharmaceutically effective amount of a pharmaceutical compositioncomprising a nucleic acid encoding a VEGF inhibitor to a human subjectin need of treatment. In some aspects, the VEGF inhibitor is ananti-VEGF antibody or a functional fragment thereof. In some aspects,the VEGF inhibitor is a soluble receptor, fusion protein, or afunctional fragment thereof.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrative aspects,in which the principles of the disclosure are utilized, and theaccompanying drawings of which:

FIG. 1 depicts the schematic representation of an exemplary plasmid.

FIG. 2 depicts expression, secretion and biological activity of sFLT-1from rAAV.sflt-1-transduced cells. (a) Western blot analysis ofconditioned media from Ad.sFlt-1-transduced 293 cells (lane 1),rAAV.sFlt-1-transduced D407 cells (lane 2), rAAV.sFlt-1-transduced 293cells (lane 3), and AAV.gfp-transduced D407 cells (lane 4). (b)Inhibition of VEGF-induced HUVEC proliferation by conditioned media fromrAAV.sFlt-1-transduced cells. HUVECs were cultured in starvation medium(column 1), in medium containing recombinant VEGF (column 2), in mediumcontaining VEGF and 40 μL conditioned medium from rAAV.sFlt-1-transduced293 cells (column 3), in medium containing VEGF and 80 μL conditionedmedium from rAAV.sFlt-1-transduced 293 cells (column 4), and in mediumcontaining VEGF and 80 μL conditioned medium from rAAV.gfp-transduced293 cells (column 5). (*P<0.02, **P<0.005 for differences betweenrAAV.sFlt-1 plus VEGF, and VEGF only.

FIG. 3A depicts graph showing human sFlt-1 (hsFLT-1) expression in thevitreous of monkeys injected in the left eyes with rAAV.sFlt-1 (Monkey8514, 8530, 8523, 8524 and 999), rAAV.gfp (Monkey 8297 and 8532), inboth eyes with recombinant sFLT-1 protein (Monkey 8294) and controluninjected monkey (control). Control and monkeys 8294 and 999 wereeuthanized at 3 months post injection, Monkey 8524 was euthanized at 9months post injection and monkeys 8297, 8532, 8514, 8530 and 8523 wereeuthanized at 12 months post injection. * denotes sFLT-1 protein levelsthat are significantly higher in the rAAV.sFlt-1 injected eyes (p<0.05).FIG. 3B depicts graphs showing hsFLT-1 levels in therAAV.sFlt-1-injected (999, 8524, 8523, 8530 and 8514), rAAV.gfp-injected(8297 and 8532), recombinant sFlt-1 protein-injected (8294) anduninjected (control) monkeys at different times post injection.

FIGS. 4A-4F depict Immune Cell Subset Populations in mouse eyesfollowing subretinal injection of rAAV-sFlt-1 at the different timespost injection. (A) Total immune cell numbers; (B) CD45 number; (C)CD11b numbers; (D) CD4 numbers; (E) CD8 numbers; (F) CD19 numbers.

FIGS. 5A-5E depict Immune Cell Subset Populations in mouse spleensfollowing subretinal injection of rAAV-sFlt-1 at the different timespost injection. (A) CD45 numbers; (B) CD 11b numbers; (C) CD4 numbers;(D) CD8 numbers; (E) CD19 numbers.

FIGS. 6A-6B depict IFN-γ production in mitogen-stimulated CD4+ and CD8+T cells from rAAV.sFlt-1 mice at different times post injection. (A)IFN-γ production in mitogen-stimulated CD4+ cells; (B) IFN-γ productionin mitogen-stimulated CD8+ cells.

FIGS. 7A to 7D depict various exemplary replication origin sequences.

FIGS. 8A to 8F depict the sequences of various exemplary promoters.

FIGS. 9A to 9C depict the sequence of various exemplary introns, poly Asequences, and ITR regions.

FIGS. 9D to 9F depict the sequence of various exemplary linkersequences.

FIGS. 9G to 9H depict the sequence of various exemplary UTR sequences.

FIGS. 10A to 10C depict the sequence encoding various exemplaryanti-VEGF proteins.

FIG. 11A depicts the amino acid sequence of sFLT-1. FIG. 11B depicts theamino acid sequence of sFLT-1 domain 2, a functional fragment of sFLT-1.FIG. 11C depicts the nucleotide sequence of VEGF inhibitor.

FIGS. 12A to 12B depict the sequences of various exemplary antibioticresistance genes.

FIG. 13 depicts the PK of one exemplary composition (rAAV.sFlt-1),wherein it reaches optimal anti-VEGF expression at 6-8 weeks. RBZ is astandard care of anti-VEGF, such as ranibizumab. “RBZ rescue” meansrescue treatment.

FIG. 14 depicts ophthalmologic assessment of the patients. Inflammationwas evaluated by biomicroscopy (BE) and indirect ophthalmoscopy (JOE).Unrem: unremarkable.

FIGS. 15A and 15B depict visual acuity results.

FIG. 16 depicts the measurement of retina thickness of a patient who wasgiven 24 previous Lucentis injections.

FIG. 17 depicts biodistribution: qPCR for sFLT-1 sequence (copy numberdetected).

FIG. 18 depicts biodistribution: AAV capsid measured by ELISA, AAV titerin capsids/mL.

FIG. 19 depicts biodistribution of sFLT-1 measured by ELISA. Shown arehuman sFLT-1 concentration (pg/mL).

FIGS. 20A and 20B depict OCT assessments of patients administered witheither low dose rAAV.sFlt-1 (R1, R2, R4) or high dose of rAAV.sFlt-1(R5, R6 and R8).

FIGS. 21A and 21B depict visual acuity results of human subjects treatedwith rAAV.sFlt-1 vs. untreated control patients at 180 days followingtreatment.

FIGS. 22A and 22B depict visual acuity results of human subjects treatedwith rAAV.sFlt-1 vs. untreated control patients at 1 year aftertreatment.

FIG. 23 depicts a table of human subjects who received Lucentis rescueinjections (VEGF inhibitor readministration) by week in a clinical studyof rAAV.sFlt-1.

FIG. 24 depicts visual acuity and SD-OCT images by week for a humansubject treated with rAAV.sFlt-1 in a clinical study of rAAV.sFlt-1.

FIGS. 25A and 25B depicts data on production of human sFlt-1 protein inhuman embryonic kidney 293 (HEK293) cells as detected by ELISA.rAAV.sFlt-1 was produced using plasmid transfection in HEK293 cells. Asecond construct, rAAV(bv).sFlt-1, was produced using recombinantbaculovirus in Sf9 insect cells. sFlt-1 protein concentration wasmeasured via ELISA after 72 at various MOI.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure provides compositions and methods for theprevention or treatment of ocular neovascularization, such as AMD, in ahuman subject, by administering subretinally a pharmaceuticalcomposition comprising a pharmaceutically effective amount of a vectorcomprising a nucleic acid encoding soluble Fms-related tyrosine kinase-1(sFlt-1) protein to the human subject.

Several aspects of the disclosure are described below with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the disclosure. One having ordinaryskill in the relevant art, however, will readily recognize that thedisclosure can be practiced without one or more of the specific detailsor with other methods. The present disclosure is not limited by theillustrated ordering of acts or events, as some acts may occur indifferent orders and/or concurrently with other acts or events.Furthermore, not all illustrated acts or events are required toimplement a methodology in accordance with the present disclosure.

The terminology of the present disclosure is for the purpose ofdescribing particular cases only and is not intended to be limiting ofcompositions, methods and compositions of this disclosure.

The compositions and methods of this disclosure as described herein mayemploy, unless otherwise indicated, conventional techniques anddescriptions of molecular biology (including recombinant techniques),cell biology, biochemistry, immunochemistry and ophthalmic techniques,which are within the skill of those who practice in the art. Suchconventional techniques include methods for observing and analyzing theretina, or vision in a subject, cloning and propagation of recombinantvirus, formulation of a pharmaceutical composition, and biochemicalpurification and immunochemistry. Specific illustrations of suitabletechniques can be had by reference to the examples herein. However,equivalent conventional procedures can, of course, also be used. Suchconventional techniques and descriptions can be found in standardlaboratory manuals such as Green, et al., Eds., Genome Analysis: ALaboratory Manual Series (Vols. I-IV) (1999); Weiner, et al., Eds.,Genetic Variation: A Laboratory Manual (2007); Dieffenbach, Dveksler,Eds., PCR Primer: A Laboratory Manual (2003); Bowtell and Sambrook, DNAMicroarrays: A Molecular Cloning Manual (2003); Mount, Bioinformatics:Sequence and Genome Analysis (2004); Sambrook and Russell, CondensedProtocols from Molecular Cloning: A Laboratory Manual (2006); andSambrook and Russell, Molecular Cloning: A Laboratory Manual (2002) (allfrom Cold Spring Harbor Laboratory Press); Stryer, L., Biochemistry (4thEd.) W.H. Freeman, N.Y. (1995); Gait, “Oligonucleotide Synthesis: APractical Approach” IRL Press, London (1984); Nelson and Cox, Lehninger,Principles of Biochemistry, 3rd Ed., W.H. Freeman Pub., New York (2000);and Berg et al., Biochemistry, 5th Ed., W.H. Freeman Pub., New York(2002), all of which are herein incorporated by reference in theirentirety for all purposes. Before the present compositions, researchtools and methods are described, it is to be understood that thisdisclosure is not limited to the specific methods, compositions, targetsand uses described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to limit thescope of the present disclosure, which will be limited only by appendedclaims.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising”.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another case includes from the one particular value and/or tothe other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another case. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. The term “about” as used herein refers to a range that is 15%plus or minus from a stated numerical value within the context of theparticular usage. For example, about 10 would include a range from 8.5to 11.5. The term “about” also accounts for typical error or imprecisionin measurement of values.

I. AMD

AMD is the leading cause of blindness in patients over the age of 50 andit is characterized by progressive degeneration of the photoreceptors,outer retina, and retinal pigment epithelium at the macula. The advanced“wet” form (neovascular or exudative) of AMD is less common, but mayfrequently cause a rapid and often substantial loss of central vision inpatients. In the wet form of AMD, choroidal neovascularization forms anddevelops into a network of vessels that may grow under and through theretinal pigment epithelium. As this is accompanied by leakage of plasmaand/or hemorrhage into the subretinal space, there could be severesudden loss of central vision if this occurs in the macula.

The term “AMD” if not otherwise specified, can be either dry AMD or wetAMD. The present disclosure contemplates treatment or prevention of AMD,wet AMD and/or dry AMD.

As is previously known in the art, AMD has been shown to have no singlecause. This highly complex disease may result from variablecontributions including but not limited to age, genetic predisposition,and environment or combination thereof. In humans, for example,established epidemiologic risk factors may include but are not limitedto cigarette smoking, diet, female sex, Caucasian race, and a familyhistory of AMD. Because AMD is rare in individuals younger than 50years, the only required risk factor is age, which implicates themultitude of cellular changes that accompany normal aging in thepathogenesis of AMD.

The etiologic complexity of AMD is reflected by the relative paucity ofeffective therapies, preventive strategies, and good animal models withwhich to study it. Due to the complexity and incomplete characterizationof the disease, AMD is incompletely modeled in animals. This is in partdue to anatomical differences in animal and primate retinas, as well asthe protracted time needed for the disease to develop. Evidence fromhuman molecular genetic and animal studies support the notion thataltered homeostasis of a multitude of mechanisms responsible for normalphotoreceptor—RPE physiology can precipitate the disease. At least onthe molecular level, the disease can be explored in animal models and,in some cases, even in those whose gene defects are not the primarycauses of AMD in humans.

Previous genetic studies as well as in depth pathological analysis,reveals that no simple inheritance pattern for AMD, and no one pathologyis common to various AMD animal models. While nonhuman primate modelsare known in the art to better approximate CNV in humans, than mice orrat models, fundamental differences in retinal anatomy, histology andeven genetics of nonhuman primates yield different species specificpathologies.

Further, and as describe herein, laser photocoagulation may be used toinduce CNV, one AMD like symptom in animal models. In some cases, lasertreatment ruptures the Bruch's membrane and evokes a fibrovascularproliferative response that originates in the choroid. This response isthe basis for modeling choroidal neovascularization in late-stage AMDand was developed in rhesus and cynomolgus macaques.

Using an argon laser, spots are kept small and induced with sufficientpower to rupture the Bruch's membrane. This is funduscopically visibleas a bubble at the time of photocoagulation. Photocoagulation inducesthrombosis of choroidal vessels followed by re-endothelialization 48hours later and growth of new vessels into the subretinal space by aweek. Because newly formed vessels are more permeable, neovasculardevelopment can be monitored with fluorescein angiography to assessvessel leakage.

Spontaneous neovascular involution (indicated by decreased fluoresceinleakage) commences at approximately 3 to 7 weeks and then graduallyprogresses (over a period of approximately 2 to 13 months) until leakageis no longer apparent at the site.

The extent of new vessel growth compared to poorly vascularized scarringcan be variable in all models and is influenced by species, location ofinjury in the retina, and intensity of the laser beam. The inherentvariability in differences of treatment from species to species furthersupports the idea that no one animal model fully recapitulates AMD inhumans.

Therapies for AMD have changed during the past few years, with theavailability of aptamers, antibodies, and soluble receptor decoys thatbind the protein VEGF. The VEGF protein or VEGF ligand, has been shownto stimulate the formation of new blood vessels (i.e. angiogenesis)through binding to cellular receptors, including the VEGF receptor. Asknown in the art, anti-VEGF agents may prevent, to some extent, theneovascularization and angiogenesis that occurs in wet AMD. Intraocularinjection of Macugen® or Lucentis® or Eylea® (anti-VEGF agents) iscostly, and in most cases the treatment must be repeated every four tosix weeks or every eight weeks in the case of Eylea®. For example,Lucentis is a VEGF antibody fragment which costs about $1950/inj.Monthly. Avastin (VEGF Antibody) is used off label, and Eylea (VEGFtrap) costs about, $1850/inj and is administered every second month. Allof these medicines share common problems of decreasing pharmacokineticprofile and thus require repeat ocular injections.

There is a need in the art for a practical, economically viable, longerlasting treatment strategy. The disclosure provides for a noveltherapeutic to address some of these needs.

The present disclosure provides an anti-VEGF molecule, such as sFLT-1,delivered by any suitable vector, (e.g. recombinant viral system) to theretina of a human subject having or suspected of having AMD or relatedneovascular retinal diseases. In some cases, sFLT-1 may be potent directbinding protein of VEGF. In some cases, sFLT-1 may also block or inhibitVEGF activity.

For example, as known in the art, sFLT-1 (as described further herein)has been observed to bind to the VEGF protein dimer with a Kd=10 pM.

The present invention also provides compositions and methods related torAAV mediated gene delivery into the eye. Long term gene expression indog eyes (>8 years) has been observed with AAV based system. sFLT-1 mRNAexpression in the retina is maintained at least for 18 months. Threehuman trials for Leber's congenital amarousis have been conducted thatdemonstrated the safety of an AAV based delivery system in the contextof a retinal degenerative disease such as LCA.

II. VEGF and Fms-Related Tyrosine Kinase-1 (sFLT-1) Protein

A. VEGF

Vascular endothelial growth factor (herein referred to as “VEGF” or“VEGF ligand”) is a potent endothelial cell-specific mitogen that playsa key role in physiological blood vessel formation. In some cases, VEGFactivity results from the binding of VEGF ligand to one or more VEGFreceptors in a cell. The binding of VEGF ligand to VEGF receptor mayhave numerous downstream cellular and biochemical effects, including butnot limited to angiogenesis in tissues. VEGF has been implicated invirtually every type of angiogenic or neovascular disorder, includingthose associated with cancer, ischemia, and inflammation. Additionally,VEGF has been implicated in eye diseases, including but not limited toischemic retinopathy, intraocular neovascularization, age-relatedmacular degeneration (AMD), wet-AMD, dry-AMD, retinalneovascularization, diabetic macular edema, diabetic retina ischemia,diabetic retinal edema, proliferative diabetic retinopathy, retinal veinocclusion, central retinal vein occlusion, branched retinal veinocclusion. Further, anti-VEGF treatments, including the compositions andmethods of this disclosure as described herein, may be used in thetreatment of one or more of these diseases described herein.

Recent data suggests that VEGF is the principal angiogenic growth factorin the pathogenesis of the wet form of AMD.

VEGF, a 46-kDa homodimeric glycopeptide, is expressed by severaldifferent ocular cell types including but not limited to pigmentepithelial cells, pericytes, vascular endothelial cells, neuroglia andganglion cells., In some cases, VEGF is express in specific spatial andtemporal patterns during retinal development. In some cases, the humanisoforms of VEGF may include proteins of 206, 189, 183, 165, 148, 145,and 121 amino acids per monomer, however the predominant human VEGFisoforms include but are not limited to VEGF121, VEGF165, VEGF189 andVEGF206. These proteins are produced by alternative splicing of the VEGFmRNA and differ in their ability to bind to heparin and to the specificVEGF receptors or coreceptors (neuropilins) The domain encoded by exons1-5 of the VEGF gene contains information required for the recognitionof the known VEGF receptors KDR/FLK-1 and FLT-1. This domain is presentin all of the VEGF isoforms. VEGF acts via these receptors, which arehigh-affinity receptor tyrosine kinases, leading to endothelial cellproliferation, migration, and increased vasopermeability.

VEGF is one of the several factors involved in the complex process ofangiogenesis and has a very high specificity for vascular endothelialcells. VEGF is a regulator of physiological angiogenesis duringprocesses such as embryogenesis, skeletal growth and reproductivefunction, but it has also been implicated in pathological angiogenesisassociated with disease such as in cancer, placental disorders and otherconditions. The potential biological effects of VEGF may be mediated byspecific fms-like membrane spanning receptors, FLT-1 and FLK-1/KDR. Insome cases, these naturally occurring binding partners of VEGF mayeffect binding of VEGF to VEGF receptors, thus modulating activation ofthe VEGF receptor and subsequent downstream pathways.

As related to cancer, several VEGF inhibitors, including a humanizedmonoclonal antibody to VEGF (rhuMab VEGF), an anti-VEGFR-2 antibody,small molecules inhibiting VEGFR-2 signal transduction and a solubleVEGF receptor have shown some therapeutic properties.

As related to intraocular neovascular diseases, such as diabeticretinopathy, retinal vein occlusions, or age related maculardegeneration, some VEGF antagonists have shown therapeutic effects,despite the need for frequent administration.

B. Anti-VEGF

The recombinant virus of the present disclosure comprises the sequenceencoding an anti-VEGF protein, including, but not limited to theVEGF-binding proteins or functional fragments thereof disclosed in U.S.Pat. Nos. 5,712,380, 5,861,484 and 7,071,159 and VEGF-binding fusionproteins disclosed in U.S. Pat. No. 7,635,474. An anti-VEGF protein mayalso include the sFLT-1 protein as described herein.

The recombinant viruses or plasmids of the present disclosure maycomprise the sequence encoding an anti-VEGF protein, including thenaturally occurring protein sFlt-1, as described in U.S. Pat. No.5,861,484 and that sequence described by SEQ ID NO: 109. It alsoincludes, but is not limited to functional fragments thereof, includingsequences of sFlt-1 domain 2 or those set forth in SEQ ID NO: 121, aswell as related constructs, such as the VEGF-binding fusion proteinsdisclosed in U.S. Pat. No. 7,635,474. An anti-VEGF protein may alsoinclude the sFLT-1 protein as described herein. These sequences can beexpressed from DNA encoding such sequences using the genetic code, astandard technique that is understood by those skilled in the art. Ascan be appreciated by those with skill in the art, due to the degeneracyof the genetic code, anti-VEGF protein sequences can be readilyexpressed from a number of different DNA sequences.

“sFlt-1 protein” herein refers to a polypeptide sequence, or functionalfragment thereof, with at least 90%, or more, homology to the naturallyoccurring human sFLT-1 sequence, such that the sFlt-1 protein orpolypeptide binds to VEGF and/or the VEGF receptor. Homology refers tothe % conservation of residues of an alignment between two sequences(e.g. as Naturally occurring human sFLT-1 protein may include anysuitable variants of sFLT-1, including, but not limited to functionalfragments, sequences comprising insertions, deletions, substitutions,pseudofragments, pseudogenes, splice variants or artificially optimizedsequences. In some cases, “sFLT-1 protein” may be at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100%homologous to the naturally occurring human sFLT-1 protein sequence. Insome cases, “sFLT-1 protein” may be at most about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% homologous to thenaturally occurring human sFLT-1 protein sequence. In some cases,“sFLT-1 protein” may be at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% spatially homologous to thenaturally occurring human sFLT-1 protein conformation. In some cases,“sFLT-1 protein” may be at most about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.9%, 99.99% or 100% spatially homologous to thenaturally occurring human sFLT-1 protein conformation.

Further, the soluble truncated form of the VEGF receptor FLT-1, sFLT-1,is the only known endogenous specific inhibitor of VEGF. In nature, itis generated by alternative mRNA splicing and lacks themembrane-proximal immunoglobulin-like domain, the transmembrane spanningregion and the intracellular tyrosine-kinase domain. Structurally, FLT-1and sFLT-1 protein may both comprise multiple functional domains. Insome variants, FLT and sFLT proteins commonly share 6 interlinkeddomain; 3 domains involved in dimerization of the protein and 3 domainsinvolved in the binding of a ligand, such as VEGF.

sFLT-1 is a soluble truncated form of the FLT-1 and it is expressedendogenously. As described herein, “soluble” FLT-1, or sFLT-1 refers toFLT-1 that is not restricted to the cellular membrane. Unbound sFLT-1may diffuse freely in extracellular space or solution.

sFLT-1 is the only known endogenous specific inhibitor of VEGF. Thisinteraction is specific and can be competed away with 100-fold excessunlabeled VEGF. In some cases, the angiostatic activity of sFLT-1 mayresult from inhibition of VEGF by two mechanisms: i) sequestration ofVEGF, to which it binds with high affinity, and ii) formation ofinactive heterodimers with membrane-spanning isoforms of the VEGFreceptors FLTt-1 and FLK-1/KDR. As known in the art, in vitro bindingassays have indicate that sFLT-1 binds VEGF with high affinity and mayalso inhibit VEGF driven proliferation of human umbilical veinendothelial cells. In animal models for cancer, sFLT-1 inhibits tumorgrowth. In some cases, sFLT-1 may function in a substoichiometric ordominant negative manner, as excess VEGF in the extracellular space maybe prevented from binding and subsequently activating the VEGF receptor.These properties of sFLT-1 have been described in Kendall and Thomas,1993; Proc Natl Acad. Sci. 90: 10705-10709, which is incorporated hereinby reference in its entirety. As is known in the art, functionalfragments of sFLT-1 can be used in place of the full-length protein.More specifically, the VEGF binding domain (domain 2), or alternativelydomain 2 of sFLT-1 plus domain 3 from sFLT1, KDR, or another familymember, can be used to bind and inactivate VEGF. Such functionalfragments are described in Wiesmann et al., 1997; Cell, 91: 695-704,which is incorporated herein by reference in its entirety. The terms“sFLT-1” and “a functional fragment of sFLT-1” are equivalent and usedhere interchangeably.

III. Vectors and Recombinant Viruses

The compositions and methods of the disclosure provide for the deliveryof a nucleic acid encoding an anti-VEGF (e.g. sFLT-1 proteins) to cellsin a human subject or patient in need thereof. In some cases, deliveryof the nucleic acid may be referred to as gene therapy.

The composition and methods of the disclosure provide for any suitablemethod for delivery of the anti-VEGF nucleic acid (e.g. sFLT-1). In somecases, delivery of the nucleic acid may be performed using any suitable“vector” (sometimes also referred to as “gene delivery” or “genetransfer vehicle). Vector, delivery vehicle, gene delivery vehicle orgene transfer vehicle, may refer to any suitable macromolecule orcomplex of molecules comprising a polynucleotide to be delivered to atarget cell. In some cases, a target cell may be any cell to which thenucleic acid or gene is delivered. The polynucleotide to be deliveredmay comprise a coding sequence of interest in gene therapy, such as thesFLT-1 gene.

For example, suitable vectors may include but are not limited to, viralvectors such as adenoviruses, adeno-associated viruses (AAV), andretroviruses, liposomes, other lipid-containing complexes, and othermacromolecular complexes capable of mediating delivery of apolynucleotide to a target cell.

In some cases, a vector may be an organic or inorganic molecule. In somecases, a vector may be small molecule (i.e. <5 kD), or a macromolecule(i.e. >5 kD). For example a vector may include but is not limited toinert, non-biologically active molecules such as metal particles. Insome cases, a vector may be gold particles.

In some cases a vector may comprise a biologically active molecule. Forexample, vectors may comprise polymerized macromolecules such asdendrimers.

In some cases, a vector may comprise a recombinant viral vector thatincorporates one or more nucleic acids. As described herein, nucleicacids may refer to polynucleotides. Nucleic acid and polynucleotide maybe used interchangeably. In some cases nucleic acids may comprise DNA orRNA. In some cases, nucleic acids may include DNA or RNA for theexpression of sFLT-1. In some cases RNA nucleic acids may include butare not limited to a transcript of a gene of interest (e.g. sFLT-1),introns, untranslated regions, termination sequences and the like. Inother cases, DNA nucleic acids may include but are not limited tosequences such as hybrid promoter gene sequences, strong constitutivepromoter sequences, the gene of interest (e.g. sFLT-1), untranslatedregions, termination sequences and the like. In some cases, acombination of DNA and RNA may be used.

As described in the disclosure herein, the term “expression construct”is meant to include any type of genetic construct containing a nucleicacid or polynucleotide coding for gene products in which part or all ofthe nucleic acid encoding sequence is capable of being transcribed. Thetranscript may be translated into a protein. In some cases it may bepartially translated or not translated. In certain aspects, expressionincludes both transcription of a gene and translation of mRNA into agene product. In other aspects, expression only includes transcriptionof the nucleic acid encoding genes of interest.

In one aspect, the present disclosure provides a recombinant virus, suchas adeno-associated virus (rAAV) as a vector to mediate the expressionof sFLT-1.

In some cases, the viral vector of the disclosure may be measured as pfu(plaque forming units). In some cases, the pfu of recombinant virus, orviral vector of the compositions and methods of the disclosure may beabout 10⁸ to about 5×10¹⁰ pfu. In some cases, recombinant viruses ofthis disclosure are at least about 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸,6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹,7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, and 5×10¹⁰ pfu. Insome cases, recombinant viruses of this disclosure are at most about1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹,2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰,3×10¹⁰, 4×10¹⁰, and 5×10¹⁰ pfu.

In some cases, the viral vector of the disclosure may be measured asvector genomes. In some cases, recombinant viruses of this disclosureare 1×10¹⁰ to 3×10¹² vector genomes. In some cases, recombinant virusesof this disclosure are 1×10⁹ to 3×10¹³ vector genomes. In some cases,recombinant viruses of this disclosure are 1×10⁸ to 3×10¹⁴ vectorgenomes. In some cases, recombinant viruses of the disclosure are atleast about 1×10¹, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷,and 1×10¹⁸ vector genomes. In some cases, recombinant viruses of thisdisclosure are 1×10⁸ to 3×10¹⁴ vector genomes. In some cases,recombinant viruses of the disclosure are at most about 1×10¹, 1×10²,1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹²,1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ vector genomes.

In some cases, the viral vector of the disclosure may be measured usingmultiplicity of infection (MOI). In some cases, MOI may refer to theratio, or multiple of vector or viral genomes to the cells to which thenucleic may be delivered. In some cases, the MOI may be 1×10⁶. In somecases, the MOI may be 1×10⁵-1×10⁷. In some cases, the MOI may be1×10⁴-1×10⁸. In some cases, recombinant viruses of the disclosure are atleast about 1×10¹, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷,and 1×10¹⁸ MOI. In some cases, recombinant viruses of this disclosureare 1×10⁸ to 3×10¹⁴ MOI. In some cases, recombinant viruses of thedisclosure are at most about 1×10¹, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶,1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵,1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ MOI.

In some aspects the nucleic acid may be delivered without the use of avirus (i.e. with a non-viral vector), and may be measured as thequantity of nucleic acid. Generally, any suitable amount of nucleic acidmay be used with the compositions and methods of this disclosure. Insome cases, nucleic acid may be at least about 1 pg, 10 pg, 100 pg, 1pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800pg, 900 pg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg,700 μg, 800 μg, 900 μg, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg,400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg 1 g, 2 g, 3 g, 4 g, or 5g. In some cases, nucleic acid may be at most about 1 pg, 10 pg, 100 pg,1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800pg, 900 pg, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg,700 μg, 800 μg, 900 μg, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg,400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, or 5g.

In some aspects, a self-complementary vector (sc) may be used. The useof self-complementary AAV vectors may bypass the requirement for viralsecond-strand DNA synthesis and may lead to greater rate of expressionof the transgene protein, as provided by Wu, Hum Gene Ther. 2007,18(2):171-82, incorporated by reference herein.

In some aspects, several AAV vectors may be generated to enableselection of the most optimal serotype, promoter, and transgene.

In some cases, the vector can be a targeted vector, especially atargeted vector that selectively binds to a specific cell, such ascancer cells or tumor cells or eye cells. Viral vectors for use in thedisclosure can include those that exhibit low toxicity to a target celland induce production of therapeutically useful quantities of theanti-VEGF protein in a cell specific manner.

The compositions and methods of the disclosure provide for any suitableviral nucleic acid delivery systems including but not limited to use ofat least one of an adeno-associated virus (AAV), adenovirus,helper-dependent adenovirus, retrovirus, herpes simplex virus,lentivirus, poxvirus, hemagglutinatin virus of Japan-liposome (HVJ)complex, Moloney murine leukemia virus, and HIV-based virus. Preferably,the viral vector comprises a strong eukaryotic promoter operably linkedto the polynucleotide e.g., a cytomegalovirus (CMV) promoter.

Generally, any suitable viral vectors may be engineered to be optimizedfor use with the compositions and methods of the disclosure. Forexample, viral vectors derived from adenovirus (Ad) or adeno-associatedvirus (AAV) may be used. Both human and non-human viral vectors can beused and the recombinant viral vector can be altered such that it may bereplication-defective in humans. Where the vector is an adenovirus, thevector can comprise a polynucleotide having a promoter operably linkedto a gene encoding the anti-VEGF protein and is replication-defective inhumans.

To combine advantageous properties of two viral vector systems, hybridviral vectors may be used to deliver a nucleic acid encoding a sFLT-1protein to a target cell or tissue. Standard techniques for theconstruction of hybrid vectors are well-known to those skilled in theart. Such techniques can be found, for example, in Sambrook, et al., InMolecular Cloning: A laboratory manual. Cold Spring Harbor, N.Y. or anynumber of laboratory manuals that discuss recombinant DNA technology.Double-stranded AAV genomes in adenoviral capsids containing acombination of AAV and adenoviral ITRs may be used to transduce cells.In another variation, an AAV vector may be placed into a “gutless”,“helper-dependent” or “high-capacity” adenoviral vector. Adenovirus/AAVhybrid vectors are discussed in Lieber et al., J. Virol. 73:9314-9324,1999. Retrovirus/adenovirus hybrid vectors are discussed in Zheng etal., Nature Biotechnol. 18:176-186, 2000.

Retroviral genomes contained within an adenovirus may integrate withinthe target cell genome and effect stable gene expression.

Replication-defective recombinant adenoviral vectors can be produced inaccordance with known techniques. See, Quantin, et al., Proc. Natl.Acad. Sci. USA, 89:2581-2584 (1992); Stratford-Perricadet, et al., J.Clin. Invest., 90:626-630 (1992); and Rosenfeld, et al., Cell,68:143-155 (1992).

Additionally preferred vectors may include but are not limited to viralvectors, fusion proteins and chemical conjugates. Retroviral vectorsinclude Moloney murine leukemia viruses and HIV-based viruses. In somecases a HIV-based viral vector may be used, wherein the HIV-based viralvector comprises at least two vectors wherein the gag and pol genes arefrom an HIV genome and the env gene is from another virus. DNA viralvectors may be used. These vectors include pox vectors such as orthopoxor avipox vectors, herpesvirus vectors such as a herpes simplex I virus(HSV) vector [Geller, A. I. et al., J. Neurochem, 64: 487 (1995); Lim,F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (OxfordUniv. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl.Acad. Sci.: U.S.A.: 90 7603 (1993); Geller, A. I., et al., Proc Natl.Acad. Sci. USA: 87:1149 (1990)], Adenovirus Vectors [LeGal LaSalle etal., Science, 259:988 (1993); Davidson, et al., Nat. Genet. 3: 219(1993); Yang, et al., J. Virol. 69: 2004 (1995)] and Adeno-associatedVirus Vectors [Kaplitt, M. G., et al., Nat. Genet. 8:148 (1994)],incorporated by reference herein.

Other viral vectors that can be used in accordance with the presentdisclosure include herpes simplex virus (HSV)-based vectors. HSV vectorsdeleted of one or more immediate early genes (IE) are advantageousbecause they are generally non-cytotoxic, persist in a state similar tolatency in the target cell, and afford efficient target celltransduction. Recombinant HSV vectors can incorporate approximately 30kb of heterologous nucleic acid.

Retroviruses, such as C-type retroviruses and lentiviruses, may also beused in the disclosure. For example, retroviral vectors may be based onmurine leukemia virus (MLV)., as provided by Hu and Pathak, Pharmacol.Rev. 52:493511, 2000 and Fong et al., Crit. Rev. Ther. Drug CarrierSyst. 17:1-60, 2000, incorporated by reference herein. MLV-based vectorsmay contain up to 8 kb of heterologous (therapeutic) DNA in place of theviral genes. The heterologous DNA may include a tissue-specific promoterand a anti-VEGF protein nucleic acid. In methods of delivery toneoplastic cells, it may also encode a ligand to a tissue specificreceptor.

Additional retroviral vectors may be used including but not limited toreplication-defective lentivirus-based vectors, including humanimmunodeficiency (HIV)-based vectors, as provided by Vigna and Naldini,J. Gene Med. 5:308-316, 2000 and Miyoshi et al., J. Virol. 72:8150-8157,1998, incorporated by reference herein. Lentiviral vectors may beadvantageous in that they are capable of infecting both activelydividing and non-dividing cells. They may also be highly efficient attransducing human epithelial cells.

Lentiviral vectors for use in the disclosure may be derived from humanand non-human (including SIV) lentiviruses. Examples of lentiviralvectors include nucleic acid sequences required for vector propagationas well as a tissue-specific promoter operably linked to an anti-VEGFprotein gene. Nucleic acid sequences may include the viral LTRs, aprimer binding site, a polypurine tract, att sites, and an encapsidationsite.

A lentiviral vector may be packaged into any suitable lentiviral capsid.The substitution of one particle protein with another from a differentvirus is referred to as “pseudotyping”. The vector capsid may containviral envelope proteins from other viruses, including murine leukemiavirus (MLV) or vesicular stomatitis virus (VSV). The use of the VSVG-protein yields a high vector titer and results in greater stability ofthe vector virus particles.

Alphavirus-based vectors, such as those made from semliki forest virus(SFV) and sindbis virus (SIN), may also be used in the disclosure. Useof alphaviruses is described in Lundstrom, K., Intervirology 43:247-257,2000 and Perri et al., Journal of Virology 74:9802-9807, 2000,incorporated by reference herein.

Recombinant, replication-defective alphavirus vectors may beadvantageous because they are capable of high-level heterologous(therapeutic) gene expression, and can infect a wide target cell range.Alphavirus replicons may be targeted to specific cell types bydisplaying on their virion surface a functional heterologous ligand orbinding domain that would allow selective binding to target cellsexpressing a cognate binding partner. Alphavirus replicons may establishlatency, and therefore long-term heterologous nucleic acid expression ina target cell. The replicons may also exhibit transient heterologousnucleic acid expression in the target cell.

Pox viral vectors may introduce a gene into the cell's cytoplasm. Avipoxvirus vectors may result in only a short term expression of the gene ornucleic acid. Adenovirus vectors, adeno-associated virus vectors andherpes simplex virus (HSV) vectors may be used with the compositions andmethods of the disclosure. The adenovirus vector may result in a shorterterm expression (e.g., less than about a month) than adeno-associatedvirus, in some aspects, and may exhibit much longer expression. Theparticular vector chosen may depend upon the target cell and thecondition being treated.

Adeno-associated viruses (AAV) are small non-enveloped single-strandedDNA viruses. They are non-pathogenic human parvoviruses and may bedependent on helper viruses, including adenovirus, herpes simplex virus,vaccinia virus and CMV, for replication. Exposure to wild-type (wt) AAVis not associated or known to cause any human pathologies and is commonin the general population, usually occurring in the first decade of lifein association with an adenoviral infection.

As described herein, “AAV” refers to Adeno-associated virus “rAAV”refers to a recombinant adeno-associated virus.

In some cases, the wild-type AAV encodes rep and cap genes. The rep geneis required for viral replication and the cap gene is required forsynthesis of capsid proteins. Through a combination of alternativetranslation start and splicing sites, the small genome may be able toexpress four rep and three cap gene products. The rep gene products andsequences in the inverted terminal repeats (145 bp ITRs, which flank thegenome) may be critical in this process. To date, 11 serotypes of AAVhave been isolated. AAV2 may be used with composition and methods of thedisclosure. The compositions and methods of the disclosure provide foruse of any suitable AAV serotype. In some aspects, the AAV is selectedfrom the group consisting of: AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rh10, and hybrids thereof.

In some aspects, the present disclosure provides a recombinant viruscomprising a nucleic acid further comprising a human form of thetruncated, soluble VEGF receptor 1 (sFLT-1) and is named rAAV.sFlt-1.The vector is a recombinant, replicative-deficient adeno-associatedviral (rAAV) vector, of serotype 2. In another aspect, the vector is arecombinant, replicative-deficient adeno-associated viral (rAAV) vector,of serotype 2 named rAAV.sFlt-1.

AAV2 is the most characterized. rAAV2 has been shown to be able tomediate long-term transgene expression in the eyes of many species ofanimals. In rats, rAAV mediated reporter gene (green fluorescentprotein) was still present at 18 months post injection. In monkeys, thesame reported gene was present at 17 months post injection. Similarly,high sFLT-1 protein levels were present in the vitreous of rAAV.sFlt-1injected monkey eyes at 15 months post injection.

rAAV.sFlt-1 has been tested in animal models for intraocular neovasculardisorders. rAAV.sFlt-1 appeared to slow the progression ofneovascularization in animal models of corneal neovascularization andretinal neovascularization. Interestingly rAAV-mediated sFlt-1 indicatedsome inhibition of neovascularization in a monkey model of choroidalneovascularization (model for the wet form of age related maculardegeneration or AMD). In this study, the presence of the rAAV.sFlt-1construct showed low levels of expression of sFLT-1 in the eyes ofmonkeys and, did not affect the well-being or retinal function of themonkeys. There is no evidence to suggest any safety issues associatedwith systemic exposure to rAAV.sFlt-1. The overall positive findings andlack of toxicity of rAAV vectors in these studies, as well as thefindings with rAAV.sFlt-1 in mammalian models of choroidalneovascularization/AMD provide extensive supporting data that the vectorhas a favorable safety profile when administered to the eye.

Despite the ability of rAAV.sFlt-1 to ameleriorate certain symptoms ofAMD in the monkey model, sFLT-1 proteins levels are unexpectedly low inthe retina. Expression levels of sFLT-1 driven by a constitutivelyactive mammalian promoter have been shown in the art to provide highlevels of protein expression in numerous cell types. While not beingbound to theory, multiple possibilities may exist for this lower thanexpected expression level. As a large multi-domain protein, sFLT-1 maybe susceptible to premature proteolyitic degradation, poor kinetics ofexpression, or non optimal sorting. With respect to the latter, as asecreted protein, sFLT-1, as expressed recombinantly in cell, enters thesecretory pathway. In retinal cells, including RPE cells, sFLT-1 may besecreted either apically or basolaterally, depending on either ER orGolgi appartis sorting of the protein. In some cases, non-optimalsorting may secrete the molecule to the undesired basolatteral membrane,thus decreasing the concentration of sFLT-1 molecules available toinhibit VEGF signaling and neovascular angiogenesis on the apicalsurface of the RPE cell layer.

Additionally, it was unknown in the art how this unexpectedly lowerlevel of sFLT-1 may affect efficacy of the drug towards treatment of theactual AMD disease in humans. While barely elevated levels in the monkeymodel showed promising signs of ameliorating symptoms of AMD, the monkeyanimal model for AMD merely serves a surrogate for AMD disease. Asdescribed herein, AMD symptoms are artificially induced (via laser) inthe retina. While this model is suitable for various analysies, theactual efficacy of the drug in the treatment of symptoms in the monkeymodel is difficult to extrapolate to treatment of disease in humans.Unexpectedly lower protein levels as generated by the rAAV.sFlt-1further increases difficulty in this assessment without experiments inhumans.

In addition, 3 clinical trials on Lebers Congenital Amaurosis (LCA) arebeing conducted in the UK and USA using the rAAV2 backbone. LCA is arare inherited eye disease that appears at birth or in the first fewmonths of life and it is characterized by nystagmus, sluggish or nopupillary responses, and severe vision loss or blindness. To date, nosafety issues have been reported following injection of the rAAV2construct into the subretinal space of 6 participants in these twotrials. Both teams involved in the clinical trials concluded that theirfindings have supported further gene therapy studies In LCA patients.

Given the apparent technical difficulties in generating substantially orsustained elevated levels of sFLT-1 in monkeys, various optimizationstrategies may be taken to address one or more of the technical issuesunderlying lower protein levels of sFlt-1 in the retina afterintroduction of rAAV.sFlt-1. In some cases, optimization strategies,including ones as provided by the composition and methods of thisdisclosure may include increasing optimizing the sFlt-1 proteinsequence, or domains, introducing control elements to direct correctsorting after expression in retinal cells, or elevating levels of sFlt-1protein to compensate for any of these possible factors. In some cases,the composition and methods of the disclosure provide for specificstrategies directed toward the latter, involving the incorporation ofspecific nucleic acid sequences directed towards improving the elevatingprotein levels in human retinans over sFlt-1 levels as observedpreviously in monkey studies. As described herein, various sequences,linkers, UTRs, introns, sFLT-1 variants or combination thereof may beused to elevate protein levels of sFlt-1 protein in the retina afterexposure to rAAV.sFlt-1.

Vectors can comprise components or functionalities that further modulategene delivery and/or gene expression, or that otherwise providebeneficial properties to the targeted cells. Such other componentsinclude, for example, components that influence binding or targeting tocells (including components that mediate cell-type or tissue-specificbinding); components that influence uptake of the vector nucleic acid bythe cell; components that influence localization of the polynucleotidewithin the cell after uptake (such as agents mediating nuclearlocalization); and components that influence expression of thepolynucleotide. Such components also might include markers, such asdetectable and/or selectable markers that can be used to detect orselect for cells that have taken up and are expressing the nucleic aciddelivered by the vector. Such components can be provided as a naturalfeature of the vector (such as the use of certain viral vectors whichhave components or functionalities mediating binding and uptake), orvectors can be modified to provide such functionalities.

Selectable markers can be positive, negative or bifunctional. Positiveselectable markers allow selection for cells carrying the marker,whereas negative selectable markers allow cells carrying the marker tobe selectively eliminated. A variety of such marker genes have beendescribed, including bifunctional (i.e., positive/negative) markers(see, e.g., Lupton, S., WO 92/08796, published May 29, 1992; and Lupton,S., WO 94/28143, published Dec. 8, 1994). Examples of negativeselectable markers may include the inclusion of resistance genes toantibiotics, such as ampicillin or kanamycin. Such marker genes canprovide an added measure of control that can be advantageous in genetherapy contexts. A large variety of such vectors are known in the artand are generally available.

In some cases, nucleic acids encoding antibiotic resistances markers mayinclude but are not limited to sequences such as SEQ ID No. 110, SEQ IDNo. 111, SEQ ID No. 112, SEQ ID No. 113 or SEQ ID No. 114.

In many of the viral vectors compatible with methods of the disclosure,one or more promoters can be included in the vector to allow more thanone heterologous gene to be expressed by the vector. Further, the vectorcan comprise a sequence which encodes a signal peptide or other moietywhich facilitates expression of the anti-VEGF protein from the targetcell.

The nucleic acid encoding a gene product may be under transcriptionalcontrol by a promoter. A “promoter”, as provided herein, refers to asuitable DNA sequence required to initiate transcription of a gene. Thephrase “under transcriptional control” means that the promoter is in thecorrect location and orientation in relation to the nucleic acid tocontrol RNA polymerase initiation and expression of the gene. In somecases, promoter may include a “strong” or constitutively activepromoter. For example, the CMV promoter may be used as known in the arta constitutively active promoter. In some cases, the CMV promoter maycomprise additional regulatory elements for promoting expression. Insome cases, the CMV promoter may comprise the initial-early CMVpromoter.

In some cases a promoter may refer to a “weak” promoter, or sequencethat yields lower levels of sFLT-1 protein than a strong promoter. Insome cases a promoter may be used such that the promoter drivesselective expression of sFLT-1. In some cases a promoter or otherregulatory elements used in combination with other sequences asdescribed herein may be used to drive selective expression of sFLT-1 inan eye cell, or eye tissue.

Additionally, “promoter”, 104 may also be used herein interchangeably torefer to any additional suitable transcriptional control modules thatmay be present around the initiation site for RNA polymerases. Thecompositions and methods of this disclosure may use any suitablepromoters and transcriptional control modules for expression of atransgene, 106. Additional transcriptional control modules may includebut are not limited to elements such as HSV thymidine kinase (tk) andSV40 early transcription units. Generally, promoters may be composed ofdiscrete functional modules, each consisting of approximately 7-20 bp ofDNA, or 20-5000 bp of DNA, and contain one or more recognition sites fortranscriptional activator or repressor proteins. The composition andmethods of the disclosure provide for any suitable regulatory sequencesor combination thereof. In some cases, these transcriptional controlmodule sequences may be referred to or identified as enhancer orrepressor sequences.

At least one module in each promoter functions to position the startsite for RNA synthesis. One example is the TATA box. Other example mayinclude some promoters that lack a TATA box, such as the promoter forthe mammalian terminal deoxynucleotidyl transferase gene and thepromoter for the SV40 late genes, a discrete element overlying the startsite itself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Generally, these are located in a region 30-110 bp upstreamof the start site, although a number of promoters may contain functionalelements downstream of the start site as well. The spacing betweenpromoter elements frequently may be flexible, so that promoter functionis preserved when elements are inverted or moved relative to oneanother. In the tk promoter for example, the spacing between promoterelements can be increased to 50 bp apart before activity begins todecline. Depending on the promoter, individual elements may position tofunction either co-operatively or independently to activatetranscription.

The compositions and methods of the disclosure provide for any suitablesequences for the control of expression of a nucleic acid sequence ofinterest in the targeted cell. Thus, where a human cell is targeted,sequences may the nucleic acid coding region may be engineered to beadjacent to and under the control of a promoter that is capable of beingexpressed in a human cell. Generally, such a promoter might includeeither a human or viral promoter.

In various aspects of the disclosure, the human cytomegalovirus (CMV)immediate early gene promoter (ie-CMV), the SV40 early promoter, theRous sarcoma virus long terminal repeat, β-actin, rat insulin promoterand glyceraldehyde-3-phosphate dehydrogenase can be used to obtain ahigh level of expression of the coding sequence of interest (e.g.sFLT-1). The use of other viral or mammalian cellular or bacterial phagepromoters which are well-known in the art to achieve expression of acoding sequence of interest is contemplated as well, provided that thelevels of expression are sufficient for a given purpose. In someaspects, prokaryotic regulatory sequences may be present in the vector,such as the T7 RNA polymerase promoter sequence. In other aspects, thevector is free from such regulatory sequences. By employing a promoterwith known properties, the level and pattern of expression of theprotein of interest following transfection or transformation can beoptimized.

Selection of a promoter that is regulated in response to specificphysiologic or synthetic signals can permit inducible expression of thegene product. For example in the case where expression of a transgene,or transgenes when a multicistronic vector is utilized, is toxic to thecells in which the vector is produced in, it may be desirable toprohibit or reduce expression of one or more of the transgenes. Examplesof transgenes that may be toxic to the producer cell line arepro-apoptotic and cytokine genes. Several inducible promoter systems areavailable for production of viral vectors where the transgene productmay be toxic. The composition and methods of the disclosure provide forany suitable combination of promoter sequence, regulatory sequences andtransgene. In some cases, a combination of sequences may result in notoxicity to the cell. In some cases, a combination of sequences mayresult in high toxicity to the cell. In some cases, a combination ofsequences may result in moderate levels of toxicity in the cell.

The ecdysone system (Invitrogen, Carlsbad, Calif.) is one such systemfor transgene expression. This system is designed to allow regulatedexpression of a gene of interest in mammalian cells. It consists of atightly regulated expression mechanism that allows little basal levelexpression of the transgene, but over 200-fold inducibility. The systemis based on the heterodimeric ecdysone receptor of Drosophila, and whenecdysone or an analog such as muristerone A binds to the receptor, thereceptor activates a promoter to turn on expression of the downstreamtransgene high levels of mRNA transcripts are attained. In this system,both monomers of the heterodimeric receptor are constitutively expressedfrom one vector, whereas the ecdysone-responsive promoter which drivesexpression of the gene of interest is on another plasmid. Engineering ofthis type of system into the gene transfer vector of interest may beused in the compositions and methods of this disclosure. Cotransfectionof plasmids containing the gene of interest and the receptor monomers inthe producer cell line would then allow for the production of the genetransfer vector without expression of a potentially toxic transgene. Atthe appropriate time, expression of the transgene could be activatedwith ecdysone or muristeron A.

In some circumstances, it may be desirable to regulate expression of atransgene in a gene therapy vector. For example, different viralpromoters with varying strengths of activity may be utilized dependingon the level of expression desired. In mammalian cells, the CMVimmediate early promoter may be used to provide strong transcriptionalactivation. Modified versions of the CMV promoter that are less potenthave also been used when reduced levels of expression of the transgeneare desired. When expression of a transgene in hematopoietic cells isdesired, retroviral promoters such as the LTRs (Long Terminal Repeat)from MLV or MMTV are often used. Other viral promoters that may be useddepending on the desired effect include SV40, RSV LTR, HIV-1 and HIV-2LTR, adenovirus promoters such as from the E1A, E2A, or MLP region, AAVLTR, cauliflower mosaic Virus, HSV-TK, and avian sarcoma virus.

In some aspects, tissue-specific promoters are used to effecttranscription in specific tissues or cells so as to reduce potentialtoxicity or undesirable effects to non-targeted tissues. For example,promoters such as the PSA, probasin, prostatic acid phosphatase orprostate-specific glandular kallikrein (hK2) may be used to target geneexpression in the prostate.

In some cases, promoters or regulatory sequence elements may be used todirect selective expression in eye cells or eye tissue. For example,promoter, sequence elements or regulatory sequences found in specificeye cell types, such as retinal pigment epithelial cells, may be used ina suitable expression construct (e.g., the RPE65 or VMD2 promoter).

The selection of appropriate promoters can be readily accomplished. Insome cases a high expression, or strong promoter may be used. An exampleof a suitable promoter is the 763-base-pair cytomegalovirus (CMV)promoter. The Rous sarcoma virus (RSV) (Davis, et al., Hum Gene Ther4:151 (1993)) and MMT promoters may also be used. Certain proteins canbe expressed using their native promoter. Other elements that canenhance expression can also be included such as an enhancer or a systemthat results in high levels of expression such as a tat gene and tarelement. This cassette can then be inserted into a vector, e.g., aplasmid vector such as, pUC19, pUC118, pBR322, or other known plasmidvectors, that includes, for example, an E. coli origin of replication.See, Sambrook, et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory press, (1989). Promoters are discussed infra.The plasmid vector may also include a selectable marker such as theβ-lactamase gene for ampicillin resistance, provided that the markerpolypeptide does not adversely affect the metabolism of the organismbeing treated. The cassette can also be bound to a nucleic acid bindingmoiety in a synthetic delivery system, such as the system disclosed inWO 95/22618, incorporated by reference herein. Generally promotersequences and/or any associated regulatory sequences may comprise aboutat least 150 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp,900 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp or 10000 bp.Promoter sequences and any associated regulatory sequences, may compriseabout at most 150 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp,800 bp, 900 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp or 10000 bp.

In some aspects, the recombinant virus or plasmid comprises a promoterselected from cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV)promoter, and MMT promoter, EF-1 alpha promoter, UB6 promoter, chickenbeta-actin promoter, CAG promoter, RPE65 promoter and opsin promoter.Generally, promoter sequences and promoter/enhancer sequences asprovided by the present disclosure may include but are not limited toany sequences selected from SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19,SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No.24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ IDNo. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, SEQID No. 34, SEQ ID No. 35, SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38,SEQ ID No. 39, SEQ ID No. 340, SEQ ID No. 41, SEQ ID No. 42, SEQ ID No.43, SEQ ID No. 44, SEQ ID No. 45, SEQ ID No. 46, and SEQ ID No. 47.

In some aspects, an antibiotic marker is used in the process forproduction of the recombinant virus. Antibiotic resistance markers maybe used to identify positive transgenic cells in the generation ofrecombinant virus. In some aspects, the antibiotic marker comprises asequence encoding an antibiotic resistance gene, such as those providedherein including but not limited to sequences shown in FIG. 8A and FIG.8B. For example markers conferring resistance may include but are notlimited to kanamycin, gentamicin, ampicillin, chloramphenicol,tetracycline, doxycycline, or hygromycin. In some aspects, theantibiotic resistance gene is a non-beta-lactam antibiotic resistancegene such as kanamycin.

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus, comprise a sequence encoding a replication originsequence, such as those provided herein. Origin of replicationsequences, generally provide sequence useful for propagating a plasmid.Generally, origin of replication sequences as provided by the presentdisclosure may include but are not limited to any sequences selectedfrom sequences as provided in FIG. 7A, FIG. 7B, FIG. 7C and FIG. 7D.

In some aspects, an origin or origin of replication sequences mayinclude but is not limited to sequences such as SEQ ID No. 1, SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7,SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12,SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, or SEQ IDNo. 17.

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus, comprise an enhancer, such as those provided herein.

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus, comprise a chimeric intron or an intron, 105, such asthose provided herein and disclosed in U.S. Pat. No. 7,635,474,incorporated by reference herein. Intron or chimeric intron may be usedinterchangeably herein. In some cases, an intron may refer to anysequence that may be transcribed but is not translated. In some cases,an intron may refer to any sequence that be transcribed and is removedfrom a mature RNA transcript in a cell. In some cases, an intron maycomprise about at least 1 bp, 50 bp, 100 bp, 150 bp, 200 bp, 300 bp, 400bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 2000 bp, 3000 bp,4000 bp or 5000 bp. In some cases, an intron may comprise may compriseabout at least 1 bp, 50 bp, 100 bp, 150 bp, 200 bp, 300 bp, 400 bp, 500bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 2000 bp, 3000 bp, 4000 bpor 5000 bp. In some cases, an intron may be about 300 bp. In some cases,an intron may be about 200-400 bp. In some cases, a chimeric intron maybe about 100-500 bp. In some cases, an intron may be about 50-200 bp. Insome cases, an intron may be either an intact naturally occurring intronor a chimeric intron.

In some aspects, an intron may include but is not limited to sequencessuch as SEQ ID No. 48, SEQ ID No. 115, SEQ ID No. 116, SEQ ID No. 117,SEQ ID No. 118, SEQ ID No. 119 or SEQ ID No. 120.

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus, comprise a poly A (polyadenylation) sequence, 107,such as those provided herein (e.g. SV40 poly A sequence.). Generally,any suitable polyA sequence may be used for the desired expression ofthe transgene (i.e. sFLT-1). For example, in some cases, the presentdisclosure provides for a sequence comprising SV40 polyA sequence, orportion of SV40 polyA sequence. In some cases, native polyA sequences asfound downstream (3′UTR) of the human sFLT-1 gene as found in humangenomic sequence may be used. In other cases, polyA sequences as founddownstream of genes other than sFLT-1 may be used. In other cases, thepresent disclosure provides for polyA sequences comprising a combinationof one or more polyA sequences or sequence elements. In some cases, nopolyA sequence is used. In some cases one or more polyA sequences may bereferred to as untranslated regions (UTRs), 3′ UTRs, or terminationsequences.

In certain aspects of the disclosure, the use of internal ribosome entrysite (IRES) or foot-mouth disease virus (FMDV) elements may be used tocreate multigene, or polycistronic, messages. IRES elements are able tobypass the ribosome scanning model of 5′ methylated Cap dependenttranslation and begin translation at internal sites. IRES elements fromtwo members of the picornavirus family (poliovirus andencephalomyocarditis) have been described, as well an IRES from amammalian message. IRES elements can be linked to heterologous openreading frames. Multiple open reading frames can be transcribedtogether, each separated by an IRES, creating polycistronic messages. Byvirtue of the IRES element, each open reading frame may be accessible toribosomes for efficient translation. Multiple genes can be efficientlyexpressed using a single promoter/enhancer to transcribe a singlemessage. An alternative system for co-expression of two proteins in genetherapy delivery vectors is the FMDV 2A system. The FMDV 2A systememploys a retroviral plasmid vector in which two genes may be linked toa nucleotide sequence encoding the 2A sequence from the picornavirusfoot-and-mouth disease virus. Transcription and translation gives riseto a bicistronic mRNA and two independent protein products.

Any heterologous open reading frame can be linked to IRES elements. Thismay include genes for secreted proteins, multi-subunit proteins, encodedby independent genes, intracellular or membrane-bound proteins andselectable markers. In this way, expression of several proteins can besimultaneously engineered into a cell with a single construct and asingle selectable marker.

A polyA sequence may comprise a length of 1-10 bp, 10-20 bp, 20-50 bp,50-100 bp, 100-500 bp, 500 bp-1 Kb, 1 Kb-2 Kb, 2 Kb-3 Kb, 3 Kb-4 Kb, 4Kb-5 Kb, 5 Kb-6 Kb, 6 Kb-7 Kb, 7 Kb-8 Kb, 8 Kb-9 Kb, and 9 Kb-10 Kb inlength. A polyA sequence may comprise a length of at least 1 bp, 2 bp, 3bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp,600 bp, 700 bp, 800 bp, 900 bp, 1 Kb, 2 Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7Kb, 8 Kb, 9 Kb, and 10 Kb in length. A polyA sequence may comprise alength of at most 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp,10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp,200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 Kb, 2Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7 Kb, 8 Kb, 9 Kb, and 10 Kb in length.

In some cases, a polyA or termination sequence may include but is notlimited to sequences such as SEQ ID No. 49, SEQ ID No. 50, SEQ ID No.51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, and SEQ ID No. 55.

Generally, polyA sequences, as provided by the present disclosure, mayinclude but are not limited to any sequences selected from PolyA Regions1-10 as provided in FIG. 9A and FIG. 9B.

In some cases, polyA sequences may be optimized for various parametersaffecting protein expression, including but not limited to mRNAhalf-life of the transgene in the cell, stability of the mRNA of thetransgene or transcriptional regulation. For example, polyA sequencesmaybe altered to increase mRNA transcript of the transgene, which mayresult in increased protein expression. In some cases, the polyAsequences maybe altered to decrease the half-life of the mRNA transcriptof the transgene, which may result in decreased protein expression.

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus, comprise a polynucleotide encoding a human sFLT-1protein or a functional fragment thereof. In some cases, the recombinantvirus and/or plasmid used to generate recombinant virus, comprises anucleic acid encoding another anti-VEGF protein or VEGF inhibitor.

In some cases, a VEGF inhibitor may include but is not limited tosequences such as SEQ ID No. 102, SEQ ID No. 103, SEQ ID No. 104, SEQ IDNo. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, or SEQ ID No.122

In some cases, nucleic acids of a VEGF inhibitor may encode forpolypeptide sequences which may include but are not limited topolypeptide sequences such as SEQ ID No. 109 or SEQ ID No. 121.

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus, comprise a regulatory nucleic acid fragment that iscapable of directing selective expression of the sFLT-1 protein in aneye cell. In some cases, eye cells may comprise retinal pigmentepithelial cells (RPE).

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus, may comprise one or more untranslated regions (UTR)or sequences. Generally, any suitable UTR sequence may be used for thedesired optimal expression of the transgene (i.e. sFLT-1). For example,in some cases, UTR regions or sequences may comprise native sequences.In some cases, UTR sequences may be sequences as found upstream (5′ UTR)or downstream (3′UTR) of the human sFLT-1 gene as found in human genomicsequence or portions thereof. In other cases, UTR sequences may comprisenon-native sequences, such as found upstream or downstream of genesother than sFLT-1 or comprise sequences further comprising a combinationof one or more UTR sequence elements as further described herein. Insome cases, only a 5′ UTR sequence is used. In some cases, only a 3′ UTRsequence is used. In some cases, no UTR sequences are used.

A UTR sequence may comprise a length of 1-10 bp, 10-20 bp, 20-50 bp,50-100 bp, 100-500 bp, 500 bp-1 Kb, 1 Kb-2 Kb, 2 Kb-3 Kb, 3 Kb-4 Kb, 4Kb-5 Kb, 5 Kb-6 Kb, 6 Kb-7 Kb, 7 Kb-8 Kb, 8 Kb-9 Kb, and 9 Kb-10 Kb inlength. A UTR sequence may comprise a length of at least 1 bp, 2 bp, 3bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp,600 bp, 700 bp, 800 bp, 900 bp, 1 Kb, 2 Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7Kb, 8 Kb, 9 Kb, and 10 Kb in length. A UTR sequence may comprise alength of at most 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp,10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp,200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 Kb, 2Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7 Kb, 8 Kb, 9 Kb, and 10 Kb in length.

Generally, UTR sequences as provided by the present disclosure mayinclude but are not limited to any sequences including but to limited toSEQ ID No. 91, SEQ ID No. 2, SEQ ID No. 92, SEQ ID No. 93, SEQ ID No.94, SEQ ID No. 95, SEQ ID No. 96, SEQ ID No. 97, SEQ ID No. 98, SEQ IDNo. 99, SEQ ID No.100, and SEQ ID No. 101.

In some cases, variations of either the 5′UTR and/or 3′UTR may beoptimized for a desired level of protein expression. In some cases,3′UTR sequences may be optimized for various parameters affectingprotein expression, including but not limited to mRNA half-life of thetransgene in the cell, stability or secondary structure of the mRNA ofthe transgene or conditional regulation (e.g. binding of various factorsto modulate translation). For example, the 3′UTR sequence maybe alteredto increase the half-life of the mRNA transcript of the transgene, whichmay result in increased protein expression. In some cases, the 3′UTRsequence maybe altered to decrease the half-life of the mRNA transcriptof the transgene, which may result in decreased protein expression.

Generally, 3′ UTRs sequences may comprise various sequence elements. Thepresent disclosure provides for 3′ UTR sequences that may include butare not limited to sequence elements such as one or more polyadenylationsignals, linker sequences, spacer sequences, SECIS elements, AU-rich orARE sequences or miRNA or RNAi binding sequences, transcriptionterminator sequences, 3′ termination sequences or variants and/orcombinations thereof.

In some cases, 5′UTR sequences may be optimized for various parametersaffecting protein expression, including but not limited to mRNAhalf-life of the transgene in the cell, stability or secondary structureof the mRNA of the transgene or transcriptional regulation. For example,the 5′UTR sequences maybe altered to increase translation efficiency ofmRNA transcript of the transgene, which may result in increased proteinexpression. In some cases, the 5′UTR sequences maybe altered to decreasetranslation efficiency of mRNA transcript of the transgene, which mayresult in decreased protein expression.

Generally, 5′ UTRs sequences may comprise various sequence elements. Thepresent disclosure provides for 5′ UTR sequences that may include butare not limited to sequence elements such as one or more ribosomebinding sites (RBS), linker sequences, spacer sequences, regulatorysequences, regulatory response elements, riboswitches, sequences thatpromote or inhibit translation initiation, regulatory sequences for mRNAtransport or variants and/or combinations thereof.

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus, may comprise one or more linker or spacer sequences.As described herein, linker sequence or spacer sequence may be usedinterchangeably. Generally, a linker sequence or spacer sequence may beany suitable sequence used to create a non-contiguous sequence betweenat least two sequence elements. For example, in one aspect of thedisclosure, a linker sequence may be found inserted between an ITR-1,108 sequence, or ITR-2, 103, and an antibiotic resistance gene sequence,106 as reflected in FIG. 1A. In another example, linker sequences may beinserted adjacent to any sequence element of the recombinant virus orthe plasmid encoding the recombinant virus including the ITR sequences,the promoter or promoter/enhancer sequences, the intron sequence, thetransgene sequence and the poly A region sequence. Generally, anysuitable linker or spacer sequence may be used to create non-contiguoussequences. For example, in some cases, linker sequences may be randomlygenerated sequence. In some cases, linker sequence may be non-specificsequence optimized to prevent formation of secondary structure orintramolecular interactions that may adversely affect proteinexpression. In some cases, linker sequences may comprise any additionalfunctional sequence elements, including but not limited to introns,regulatory sequences, enhancers or the like. Functional elements inlinker sequences may be used for the desired optimal production of virusand/or expression of transgene expression. In some cases, linkersequences are cloning sites, remnants of prior cloning sites or othernon-significant sequences and the insertion of such linkers between anytwo sequence elements is optional.

Generally, linker sequence, as provided by the present disclosure, mayinclude but are not limited to any sequences selected from sequences asprovided in FIG. 9D, FIG. 9E and FIG. 9F.

In some cases, the length of the linker sequence may be optimized forthe desired optimal production of virus and/or expression of transgeneexpression. In some cases, the length of one or more linker sequenceslocated at one or more sites in the virus genome or plasmid may bevaried to produce the desired optimal protein expression. For example, alinker sequence may be found between the intron, as described herein andthe transgene (i.e. sFLT-1). The length of the linker sequence may bevaried to produce varying effects on the transcription and subsequenttranslation of the transgene in the cell.

A linker sequence may comprise a length of 1-10 bp, 10-20 bp, 20-50 bp,50-100 bp, 100-500 bp, 500 bp-1 Kb, 1 Kb-2 Kb, 2 Kb-3 Kb, 3 Kb-4 Kb, 4Kb-5 Kb, 5 Kb-6 Kb, 6 Kb-7 Kb, 7 Kb-8 Kb, 8 Kb-9 Kb, and 9 Kb-10 Kb inlength. A linker sequence may comprise a length of at least 1 bp, 2 bp,3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp,600 bp, 700 bp, 800 bp, 900 bp, 1 Kb, 2 Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7Kb, 8 Kb, 9 Kb, and 10 Kb in length. A linker sequence may comprise alength of at most 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp,10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp,200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 Kb, 2Kb, 3 Kb, 4 Kb, 5 Kb, 6 Kb, 7 Kb, 8 Kb, 9 Kb, and 10 Kb in length.

In some cases, a linker or spacer sequence may include but is notlimited to SEQ ID No. 60, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 63,SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No.68, SEQ ID No. 69, SEQ ID No.70, SEQ ID No. 71, SEQ ID No. 72, SEQ IDNo. 73, SEQ ID No. 74, SEQ ID No. 75, SEQ ID No. 76, SEQ ID No. 77, SEQID No. 78, SEQ ID No. 79, SEQ ID No. 80, SEQ ID No. 81, SEQ ID No. 82,SEQ ID No. 83, SEQ ID No. 84, SEQ ID No. 85, SEQ ID No. 86, SEQ ID No.87, SEQ ID No. 88, SEQ ID No. 89, and SEQ ID No. 90.

In some aspects, the recombinant virus comprises inverted terminalrepeat (ITR) sequences used for packaging the recombinant geneexpression cassette into the virion of the viral vector. In some cases,the ITR is from adeno-associated virus (AAV). In some cases, the ITR isfrom AAV serotype 2. In some cases, an ITR may include but is notlimited to SEQ ID No. 56, SEQ ID No. 57, SEQ ID No. 58, or SEQ ID No.59.

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus comprises nucleic acid elements in the followingorder: a) a first ITR sequence; b) a promoter sequence; c) an intronsequence; d) a first UTR sequence; e) a sequence encoding a VEGFinhibitor; f) a second UTR sequence; g) a poly A sequence; and h) asecond ITR sequence. In some aspects of the recombinant virus and/orplasmid used to generate the recombinant virus, the promoter sequencecomprises a promoter/enhancer sequence. In some aspects, the sequenceencoding a VEGF inhibitor comprises a sequence encoding human sFLT-1protein or a functional fragment thereof. In other aspects, the plasmidused to generate the recombinant virus further comprises an origin ofreplication sequence, 102. In some aspects, the plasmid furthercomprises a sequence for an antibiotic resistance gene as providedherein.

In some aspects, the recombinant virus and/or plasmid used to generaterecombinant virus comprises nucleic acid elements in the followingorder: a) a first ITR sequence; b) a first linker sequence; c) apromoter sequence; d) a second linker sequence; e) an intron sequence;f) a third linker sequence; g) a first UTR sequence; h) a sequenceencoding a VEGF inhibitor; i) a second UTR sequence; j) a fourth linkersequence; k) a poly A sequence; l) a fifth linker sequence; and m) asecond ITR sequence. In some aspects of the recombinant virus and/orplasmid used to generate recombinant virus, the promoter sequencecomprises a promoter/enhancer sequence. In some aspects, the sequenceencoding a VEGF inhibitor comprises a sequence encoding human sFLT-1protein or a functional fragment thereof. In other aspects, the plasmidused to generate the recombinant virus further comprises an origin ofreplication sequence. In some aspects, the plasmid further comprises asequence for an antibiotic resistance gene as provided herein.

IV. Pharmaceutical Compositions

A pharmaceutical composition is a formulation containing one or moreactive ingredients as well as one or more excipients, carriers,stabilizers or bulking agents, which is suitable for administration to ahuman patient to achieve a desired diagnostic result or therapeutic orprophylactic effect. For storage stability and convenience of handling,a pharmaceutical composition can be formulated as a lyophilized (i.e.freeze dried) or vacuum dried powder which can be reconstituted withsaline or water prior to administration to a patient. Alternately, thepharmaceutical composition can be formulated as an aqueous solution. Apharmaceutical composition can contain a proteinaceous activeingredient. Unfortunately, proteins can be very difficult to stabilize,resulting in loss of protein and/or loss of protein activity during theformulation, reconstitution (if required) and during the storage priorto use of a protein containing pharmaceutical composition. Stabilityproblems can occur because of protein denaturation, degradation,dimerization, and/or polymerization. Various excipients, such as albuminand gelatin have been used with differing degrees of success to try andstabilize a protein active ingredient present in a pharmaceuticalcomposition. Additionally, cryoprotectants such as alcohols have beenused to reduce protein denaturation under the freezing conditions oflyophilization.

Pharmaceutical compositions suitable for internal use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, or phosphate buffered saline(PBS). In all cases, the composition must be sterile and should be fluidto the extent that easy syringability exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants such as polysorbates (Tween™), sodium dodecyl sulfate(sodium lauryl sulfate), lauryl dimethyl amine oxide,cetyltrimethylammonium bromide (CTAB), polyethoxylated alcohols,polyoxyethylene sorbitan, octoxynol (Triton X100™),N,N-dimethyldodecylamine-N-oxide, hexadecyltrimethylammonium bromide(HTAB), polyoxyl 10 lauryl ether, Brij 721™, bile salts (sodiumdeoxycholate, sodium cholate), pluronic acids (F-68, F-127), polyoxylcastor oil (Cremophor™) nonylphenol ethoxylate (Tergitol™),cyclodextrins and, ethylbenzethonium chloride (Hyamine™) Prevention ofthe action of microorganisms can be achieved by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as manitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the internal compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile solutions can be prepared by incorporating the active compoundin the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

In one aspect, active compounds are prepared with carriers that willprotect the compound against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations will be apparent to those skilled in the art. Thematerials can also be obtained commercially. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811, incorporated by reference herein.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the humansubject to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The pharmaceutical compositions of the disclosure encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal comprising ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the disclosure, pharmaceutically acceptablesalts of such prodrugs, and other bio-equivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions.

The term “pharmaceutically acceptable salt” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of thedisclosure: i.e., salts that retain the desired biological activity ofthe parent compound and do not impart undesired toxicological effectsthereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Metals used as cations comprise sodium, potassium, magnesium, calcium,and the like. Amines comprise N—N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, dicyclohexylamine,ethylenediamine, N-methylglucamine, and procaine (see, for example,Berge et al., “Pharmaceutical Salts,” J. Pharma Sci., 1977, 66, 119).The base addition salts of said acidic compounds are prepared bycontacting the free acid form with a sufficient amount of the desiredbase to produce the salt in the conventional manner. The free acid formmay be regenerated by contacting the salt form with an acid andisolating the free acid in the conventional manner. The free acid formsdiffer from their respective salt forms somewhat in certain physicalproperties such as solubility in polar solvents, but otherwise the saltsare equivalent to their respective free acid for purposes of the presentdisclosure.

As used herein, a “pharmaceutical addition salt” comprises apharmaceutically acceptable salt of an acid form of one of thecomponents of the compositions of the disclosure. These comprise organicor inorganic acid salts of the amines. Preferred acid salts are thehydrochlorides, acetates, salicylates, nitrates and phosphates. Othersuitable pharmaceutically acceptable salts are well known to thoseskilled in the art and comprise basic salts of a variety of inorganicand organic acids, such as, for example, with inorganic acids, such asfor example hydrochloric acid, hydrobromic acid, sulfuric acid orphosphoric acid; with organic carboxylic, sulfonic, sulfo or phosphoacids or N-substituted sulfamic acids, for example acetic acid,propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleicacid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lacticacid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citricacid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin Nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfoic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and comprise alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.For oligonucleotides, preferred examples of pharmaceutically acceptablesalts comprise but are not limited to: (I) salts formed with cationssuch as sodium, potassium, ammonium, magnesium, calcium, polyamides suchas spermine and spermidine, and the like; (II) acid addition saltsformed with inorganic acids, for example hydrochloric acid, hydrobromicacid, sulfuric acid, phosphoric acid, nitric acid and the like; (III)salts formed with organic acids such as, for example, acetic acid,oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid,gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid,tannic acid, palmitic acid, alginic acid, polyglutamic acid,napthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and(IV) salts formed from elemental anions such as chlorine, bromine, andiodine.

Pharmaceutical compositions of the present disclosure comprise, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that comprise, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

Certain compositions of the present disclosure also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The co-administration of a nucleic acid and a carriercompound, generally with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extra circulatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is co-administered with polyinosinic acid, dextran sulphate,polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

The vector or recombinant viruses (virions) can be incorporated intopharmaceutical compositions for administration to mammalian patients,particularly humans. The vector or virions can be formulated innontoxic, inert, pharmaceutically acceptable aqueous carriers,preferably at a pH ranging from 3 to 8, more preferably ranging from 6to 8. Such sterile compositions will comprise the vector or virioncontaining the nucleic acid encoding the therapeutic molecule dissolvedin an aqueous buffer having an acceptable pH upon reconstitution.

In some aspects, the pharmaceutical composition provided herein comprisea therapeutically effective amount of a vector or virion in admixturewith a pharmaceutically acceptable carrier and/or excipient, for examplesaline, phosphate buffered saline, phosphate and amino acids, polymers,polyols, sugar, buffers, preservatives and other proteins. Exemplaryamino acids, polymers and sugars and the like are octylphenoxypolyethoxy ethanol compounds, polyethylene glycol monostearatecompounds, polyoxyethylene sorbitan fatty acid esters, sucrose,fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol,inositol, galactitol, xylitol, lactose, trehalose, bovine or human serumalbumin, citrate, acetate, Ringer's and Hank's solutions, cysteine,arginine, carnitine, alanine, glycine, lysine, valine, leucine,polyvinylpyrrolidone, polyethylene and glycol. Preferably, thisformulation is stable for at least six months at 4° C.

In some aspects, the pharmaceutical composition provided hereincomprises a buffer, such as phosphate buffered saline (PBS) or sodiumphosphate/sodium sulfate, tris buffer, glycine buffer, sterile water andother buffers known to the ordinarily skilled artisan such as thosedescribed by Good et al. (1966) Biochemistry 5:467. The pH of the bufferin which the pharmaceutical composition comprising the anti-VEGFcontained in the adenoviral vector delivery system, may be in the rangeof 6.5 to 7.75, 7 to 7.5, or 7.2 to 7.4. The pH of the formulation mayrange from about 3.0 to about 12.0. The pH of the immunogeniccomposition may be at least about 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 pHunits. The pH of the immunogenic composition may be at most about 3, 4,5, 6, 7, 8, 9, 10, 11 or 12 pH units.

In some aspects, the pharmaceutical composition provided hereincomprises substances which increase the viscosity of the suspension,such as sodium carboxymethyl cellulose, sorbitol, or dextran, in theamount about 1-10 percent, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10percent.

Certain aspects of the disclosure provide pharmaceutical compositionscontaining one or more recombinant virus and one or more otherchemotherapeutic agents.

Examples of such chemotherapeutic agents comprise, but are not limitedto, anticancer drugs such as daunorubicin, dactinomycin, doxorubicin,bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MIX),colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 1206-1228).

Anti-inflammatory drugs, comprising but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,comprising but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the disclosure (TheMerck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds.,1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Othernon-antisense chemotherapeutic agents are also within the scope of thisdisclosure. Two or more combined compounds may be used together orsequentially.

In another related aspect, compositions of the disclosure may containone or more recombinant viruses, particularly sFLT-1 with differentsequences. Two or more combined viruses may be used together orsequentially.

In another aspect, the present disclosure provides a unit dose of apharmaceutical composition comprising about 1×10⁶ about 1×10¹⁵ viralgenomes, wherein the viruses comprises a nucleic acid encoding sFLT-1.

In some cases, the unit dose of the pharmaceutical composition of thedisclosure may be measured as pfu (plaque forming units). In some cases,the pfu of the unit dose of the pharmaceutical composition of thedisclosure may be about 1×10⁸ to about 5×10¹⁰ pfu. In some cases, thepfu of the unit dose of the pharmaceutical composition of the disclosureis at least about 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸, 7×10⁸,8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹,9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, and 5×10¹⁰ pfu. In some cases,the pfu of the unit dose of the pharmaceutical composition of thedisclosure is at most about 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸, 5×10⁸, 6×10⁸,7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹,8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, and 5×10¹⁰ pfu.

In some cases, the viral vector of the disclosure may be measured asvector genomes. In some cases, the unit dose of the pharmaceuticalcomposition of the disclosure is 1×10¹⁰ to 3×10¹² vector genomes. Insome cases, the unit dose of the pharmaceutical composition of thedisclosure is 1×10⁹ to 3×10¹³ vector genomes. In some cases, the unitdose of the pharmaceutical composition of the disclosure is 1×10¹⁰ to1×10¹¹ vector genomes. In some cases, the unit dose of thepharmaceutical composition of the disclosure is 1×10⁸ to 3×10¹⁴ vectorgenomes. In some cases, the unit dose of the pharmaceutical compositionof the disclosure is at least about 1×10¹, 1×10², 1×10³, 1×10⁴, 1×10⁵,1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴,1×10¹⁵, 1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ vector genomes. In some cases, theunit dose of the pharmaceutical composition of the disclosure is 1×10⁸to 3×10¹⁴ vector genomes. In some cases, the unit dose of thepharmaceutical composition of the disclosure is at most about 1×10¹,1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹,1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ vectorgenomes.

In some cases, the unit dose of the pharmaceutical composition of thedisclosure may be measured using multiplicity of infection (MOI). Insome cases, MOI may refer to the ratio, or multiple of vector or viralgenomes to the cells to which the nucleic may be delivered. In somecases, the MOI may be 1×10⁶. In some cases, the MOI may be 1×10⁵-1×10⁷.In some cases, the MOI may be 1×10⁴-1×10⁸. In some cases, recombinantviruses of the disclosure are at least about 1×10¹, 1×10², 1×10³, 1×10⁴,1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³,1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ MOI. In some cases,recombinant viruses of this disclosure are 1×10⁸ to 3×10¹⁴ MOI. In somecases, recombinant viruses of the disclosure are at most about 1×10¹,1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹,1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵, 1×10¹⁶, 1×10¹⁷, and 1×10¹⁸ MOI.

In some aspects the nucleic acid may be delivered without the use of avirus (i.e. with a non-viral vector), and may be measured as thequantity of nucleic acid. Generally, any suitable amount of nucleic acidmay be used with the compositions and methods of this disclosure. Insome cases, the amount of nucleic acid may be at least about 1 pg, 10pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg,700 pg, 800 pg, 900 pg, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 μg, 10 μg, 100 μg, 200 μg, 300 μg,400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 10 mg, 100 mg, 200mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg 1 g, 2 g, 3g, 4 g, or 5 g. In some cases, nucleic acid may be at most about 1 pg,10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600pg, 700 pg, 800 pg, 900 pg, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng,500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 μg, 10 μg, 100 μg, 200 μg, 300μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 10 mg, 100 mg,200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2g, 3 g, 4 g, or 5 g.

In some aspects, the pharmaceutical composition comprises about 1×10⁶ toabout 1×10¹⁵ recombinant viruses, about 1×10⁷ to about 1×10¹⁴recombinant viruses, about 1×10⁸ to about 1×10¹³ recombinant viruses,about 1×10⁹ to about 3×10¹² recombinant viruses, or about 1×10¹⁰ toabout 3×10¹² recombinant viruses.

Kits

Compositions and reagents useful for the present disclosure may bepackaged in kits to facilitate application of the present disclosure. Insome aspects, the present method provides for a kit comprising arecombinant nucleic acid of the disclosure. In some aspects, the presentmethod provides for a kit comprising a recombinant virus of thedisclosure. The instructions could be in any desired form, including butnot limited to, printed on a kit insert, printed on one or morecontainers, as well as electronically stored instructions provided on anelectronic storage medium, such as a computer readable storage medium.Also optionally included is a software package on a computer readablestorage medium that permits the user to integrate the information andcalculate a control dose.

In another aspect, the present disclosure provides a kit comprising thepharmaceutical compositions provided herein. In yet another aspect, thedisclosure provides kits in the treatment of diseases such as, forexample: AMD, DME, RVO, angiogenesis related diseases, cancer,autoimmune diseases, infectious disease organisms, and the like.

In one aspect, a kit comprises: (a) a recombinant virus provided herein,and (b) instructions to administer to cells or an individual atherapeutically effective amount of the recombinant virus. In someaspects, the kit may comprise pharmaceutically acceptable salts orsolutions for administering the recombinant virus. Optionally, the kitcan further comprise instructions for suitable operational parameters inthe form of a label or a separate insert. For example, the kit may havestandard instructions informing a physician or laboratory technician toprepare a dose of recombinant virus.

Optionally, the kit may further comprise a standard or controlinformation so that a patient sample can be compared with the controlinformation standard to determine if the test amount of recombinantvirus is a therapeutic amount consistent with for example, a shrinkingof a tumor. Optionally, the kit could further comprise devices foradministration, such as a syringe, filter needle, extension tubing,cannula, and subretinal injector.

Recombinant viruses may be generated by any suitable means. The methodsand compositions and of the disclosure provide for generation ofrecombinant virus through various means, including the use of transgeniccells, which may include mammalian cells, insect cells, animal cells orfungal cells.

For example, in some aspects, recombinant viruses may be generatedthrough transfection of insect cells via recombinant baculovirus. Insome cases, recombinant baculovirus may be generated as an intermediate,whereby the baculovirus may contain sequences necessary for thegeneration of other viruses such as AAV or rAAV2 viruses. In some casesone or more baculoviruses may be used in the generation of recombinantviruses used for the composition and methods of treatment of thisdisclosure. In some cases insect cells such as Sf9, High-Five or Sf21cell lines may be used. In some cases, cell lines may be generated usingtransient methods (i.e. infection with not stably integratedtransgenes.) In other cases, cell lines may be generated through thegeneration of stable cell lines ((i.e. infection with transgenes stablyintegrated into the host cell genome.) In other aspects, thepharmaceutical composition provided herein is manufactured usingadherent human embryonic kidney 293 (HEK293) cells. In an alternativeaspect, the pharmaceutical composition provided herein is manufacturedusing suspension-adapted HEK293 cells. In another aspect, thepharmaceutical composition provided herein is manufactured using thebaculovirus expression system (BYES) in insect cells. In some aspects,the vector is produced using herpes-helper virus. In some aspects, thevector is produced using producer-clone methods. In some aspects, thevector is produced using Ad-AAV.

Generally, any suitable method may be used in the biochemicalpurification of recombinant viruses for use in a pharmaceuticalcomposition as described herein. Recombinant viruses may be harvesteddirectly from cells, or from the culture media surrounding host cells.Virus may be purified using various biochemical means, such as gelfiltration, filtration, chromatography, affinity purification, gradientultracentrifugation, or size exclusion methods. Recombinant virus may betested for content (i.e., identity), purity, or potency (i.e., activity)using any suitable means, before formulation into a pharmaceuticalcomposition. Method may include but are not limited to immunoassays,ELISA, SDS-PAGE, western blot, Northern blot, Southern blot or PCR,HUVEC assays and the like.

V. Method of Treatment

In another aspect, the present disclosure provided a method for treatinga pathological angiogenesis related disease, comprising administering apharmaceutically effective amount of the pharmaceutical compositionsprovided herein to a human subject in need of such treatment. In someaspects, the disease is selected from the group of ocular neovasculardiseases consisting of: age-related macular degeneration (AMD), wet-AMD,dry-AMD, retinal neovascularization, choroidal neovascularizationdiabetic retinopathy, proliferative diabetic retinopathy, retinal veinocclusion, central retinal vein occlusion, branched retinal veinocclusion, diabetic macular edema, diabetic retinal ischemia, ischemicretinopathy and diabetic retinal edema.

In some cases, dry AMD may be treated. In some cases, dry AMD may bereferred to as central geographic atrophy, characterized by atrophy ofthe retinal pigment epithelial later below the retina and subsequentloss of photoreceptors in the central part of the eye. The compositionand methods of this disclosure provide for the treatment of any and allforms of AMD.

In another aspect, the present disclosure provides a method forprophylactic treatment of AMD or ocular neovascular diseases asdescribed herein, comprising administering a pharmaceutically effectiveamount of the pharmaceutical compositions provided herein to a humansubject in need of such treatment. The present disclosure may be used totreat patients at risk of developing AMD, or presenting early symptomsof the disease. This may include treatment of eyes either simultaneouslyor sequentially. Simultaneous treatment may mean that the treatment isadministered to each eye at the same time or that both eyes are treatedduring the same visit to a treating physician or other healthcareprovider. It has been documented that patients have a higher risk ofdeveloping AMD in a healthy fellow eye of an eye that presents symptomsof AMD, or in patients who have a genetic predisposition towarddeveloping AMD. The present disclosure can be used as a prophylactictreatment in prevention of AMD in the fellow eye.

While the mechanism underlying the increased risk for the progression ofocular neovascular disease in a fellow eye is unknown, there aremultiple studies in the art detailing this elevated risk. For example,in one such large scale study, of 110 fellow eyes observed thatprogressed to advanced AMD, choroidal neovascularization (CNV) developedin 98 eyes and foveal geographic atrophy (GA) in 15 eyes.Ophthalmoligica, 2011; 226(3):110-8. doi: 10.1159/000329473. Curr OpinOphthalmol. 1998 June; 9(3):38-46. No non-ocular characteristic (age,gender, history of hypertension or smoking) or ocular feature of thestudy eye at baseline (lesion composition, lesion size, or visualacuity) was predictive of progression to advanced AMD in this cohort.However, statistical analysis indicates that AMD symptoms of the firsteye, including drusen size, focal hyperpigmentation, and nonfovealgeographic atrophy had significant independent relationships inassessing risk of developing of AMD in the fellow eye. Recent studieshave indicated that of ocular characteristics, genetic factors andcertain environmental factors may play a role in the increased risk ofdeveloping AMD in the fellow eye. JAMA Ophthalmol. 2013 Apr. 1;131(4):448-55. doi: 10.1001/jamaophthalmol.2013.2578. Given the wellcharacterized elevated risk of AMD development in untreated fellow eyes,there is need in the art of methods for preventing onset and subsequentvision loss due to the disease.

The term “subject,” or “individual” or “patient” as used herein inreference to individuals having a disease or disorder or are suspectedof having a disease or disorder, and the like. Subject, individual orpatent may be used interchangeably in the disclosure and encompassmammals and non-mammals. Examples of mammals include, but are notlimited to, any member of the Mammalian class: humans, non-humanprimates such as chimpanzees, and other apes and monkey species; farmanimals such as cattle, horses, sheep, goats, swine; domestic animalssuch as rabbits, dogs, and cats; laboratory animals including rodents,such as rats, mice and guinea pigs, and the like. Examples ofnon-mammals include, but are not limited to, birds, fish and the like.In some aspects of the methods and compositions provided herein, themammal is a human.

The term “subject” or “individual” also includes humans suffering fromthe disorder or disease, age 20 and older. Unexpectedly, the presentdisclosure can be used in a range of patient ages. This includes youngerpatients not generally associated with AMD disease, which presents morefrequently in patients over the age of 65. Human subjects, or patientsof the disclosure may include ages at least about 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100. Human subjects, orpatients of the disclosure may include ages at most about 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100.

In some aspects, the term “subject,” or “individual” includes patientswith varying responses to penicillin, such as resistance or sensitivityto its effects or patients who show or lack symptoms of allergicresponse to the drug.

A. Method of Delivery

In some aspects, the pharmaceutical composition is administered tosubretinal sites using any direction method. In some cases, the deliverymethod may be by injection, such as those described in US Pat Pub. No.2010008170, which is incorporated by reference in its entirety. In somecases, direct administration to subretinal sites includes injection of aliquid pharmaceutical composition via syringe. In another example,direct administration may involve injection via a cannula or othersuitable instrument for delivery for a vector or recombinant virus. Inother examples, direct administration may comprise an implant furthercomprising a suitable vector for delivery of transgenes such as sFLT-1.In some cases the implant may be either directly implanted in or nearthe retina.

The central retina, macula, and fovea regions of the retina are uniqueamongst mammals to primates. Furthermore, there are distinct differencesin the anatomy and subsequent pathogenesis of AMD between primate andhumans. The central retina is the area of the retina surrounding theposterior pole between the vascular arcades of a primate eye, whichincludes the fovea, macula, and surrounding area. The macula is near thecenter of the retina and has a diameter of approximately 1.5 mm. Thisarea contains the highest concentration of both rod and conephotoreceptors. At the center of the macula is the fovea, a small pitthat contains the largest concentration of cone photoreceptors. Themacula and fovea regions of the retina also contain underlying RPEcells. These regions of the retina are responsible for perception offine detail (acuity) and color. As this region is responsible for themost important part of human vision (fine vision), safe and effectivetargeting of the vector to the subretinal space of the macula and foveais desired. In some cases, a pharmaceutical composition of thedisclosure is administered in the central retina. In some cases, it isadministered in the central retina outside the fovea.

Briefly, the general method for delivering a vector to the subretinalspace of the macula and fovea may be illustrated by the following briefoutline. This example is merely meant to illustrate certain features ofthe method, and is in no way meant to be limiting.

Generally, the vector can be delivered in the form of a suspensioninjected intraocularly (subretinally) under direct observation using anoperating microscope. This procedure may involve vitrectomy followed byinjection of vector suspension using a fine cannula through one or moresmall retinotomies into the subretinal space.

Briefly, an infusion cannula can be sutured in place to maintain anormal globe volume by infusion (of e.g. saline) throughout theoperation. A vitrectomy is performed using a cannula of appropriate boresize (for example 20 to 27 gauge), wherein the volume of vitreous gelthat is removed is replaced by infusion of saline or other isotonicsolution from the infusion cannula. The vitrectomy is advantageouslyperformed because (1) the removal of its cortex (the posterior hyaloidmembrane) facilitates penetration of the retina by the cannula; (2) itsremoval and replacement with fluid (e.g. saline) creates space toaccommodate the intraocular injection of vector, and (3) its controlledremoval reduces the possibility of retinal tears and unplanned retinaldetachment.

In some aspects, the vector is directly injected into the subretinalspace within the central retina, by utilizing a cannula of theappropriate bore size (e.g. 27-45 gauge), thus creating a bleb in thesubretinal space. In other aspects, the subretinal injection of vectorsuspension is preceded by subretinal injection of a small volume (e.g.about 0.1 to about 0.5 ml) of an appropriate fluid (such as saline orRinger's solution) into the subretinal space within the central retina.This initial injection into the subretinal space establishes an initialfluid bleb within the subretinal space, causing localized retinaldetachment at the location of the initial bleb. This initial fluid blebcan facilitate targeted delivery of vector suspension to the subretinalspace (by defining the plane of injection prior to vector delivery), andminimize possible vector administration into the choroid and thepossibility of vector injection or reflux into the vitreous cavity. Insome aspects, this initial fluid bleb can be further injected withfluids comprising one or more vector suspensions and/or one or moreadditional therapeutic agents by administration of these fluids directlyto the initial fluid bleb with either the same or additional fine borecannulas.

Intraocular administration of the vector suspension and/or the initialsmall volume of fluid can be performed using a fine bore cannula (e.g.27-45 gauge) attached to a syringe. In some aspects, the plunger of thissyringe may be driven by a mechanized device, such as by depression of afoot pedal. The fine bore cannula is advanced through the sclerotomy,across the vitreous cavity and into the retina at a site pre-determinedin each subject according to the area of retina to be targeted (withinthe central retina). In one aspect, administration is performed to asite outside the fovea. Under direct visualization the vector suspensionis injected mechanically under the neurosensory retina causing alocalized retinal detachment with a self-sealing non-expandingretinotomy. As noted above, the vector can be either directly injectedinto the subretinal space creating a bleb within the central retina orthe vector can be injected into an initial bleb within the centralretina, causing it to expand (and expanding the area of retinaldetachment). In some aspects, the injection of vector suspension isfollowed by injection of another fluid into the bleb.

Without wishing to be bound by theory, the rate and location of thesubretinal injection(s) can result in localized shear forces that candamage the macula, fovea and/or underlying RPE cells. The subretinalinjections may be performed at a rate that minimizes or avoids shearforces. In some aspects, the vector is injected over about 15-17minutes. In some aspects, the vector is injected over about 17-20minutes. In some aspects, the vector is injected over about 20-22minutes. In some aspects, the vector is injected over about 1 minute orover about 1-3 minutes or in less than one minute. In some aspects, thevector is injected at a rate of about 35 to about 65 μl/min or 65 μl/minto about 150 μl/min. In some aspects, the vector is injected at a rateof about 35 μl/min. In some aspects, the vector is injected at a rate ofabout 40 μl/min. In some aspects, the vector is injected at a rate ofabout 45 μl/min. In some aspects, the vector is injected at a rate ofabout 50 μl/ml. In some aspects, the vector is injected at a rate ofabout 55 μl/min. In some aspects, the vector is injected at a rate ofabout 60 μl/ml. In some aspects, the vector is injected at a rate ofabout 65 μl/min. In some aspects, the vector is injected at a rate ofabout 100 μl/min. One of ordinary skill in the art would recognize thatthe rate and time of injection of the bleb may be directed by, forexample, the volume of the vector or size of the bleb necessary tocreate sufficient retinal detachment to access the cells of centralretina, the size of the cannula used to deliver the vector, and theability to safely maintain the position of the cannula of thedisclosure.

One or multiple (e.g. 2, 3, or more) blebs can be created. Generally,the total volume of bleb or blebs created by the methods and systems ofthe disclosure cannot exceed the fluid volume of the eye, for exampleabout 4 ml in a typical human subject. The total volume of eachindividual bleb is preferably at about 0.1-0.2 ml. One of ordinary skillin the art will appreciate that in creating the bleb according to themethods and systems of the disclosure that the appropriate intraocularpressure must be maintained in order to avoid damage to the ocularstructures. The size of each individual bleb may be, for example, about50 μl to about 100 μl, about 50 μl to about 200 μl, about 0.1 to about0.2 ml, about 0.1 to about 0.3 ml, or >0.3 ml.

In order to safely and efficiently transduce areas of target retina(e.g. the central retina) outside the edge of the original location ofthe bleb, in some cases it may be desirable to manipulate the bleb toreposition the bleb to the target area for transduction. Manipulation ofthe bleb can occur by the dependency of the bleb that is created by thevolume of the bleb, repositioning of the eye containing the bleb,repositioning of the head of the human with an eye or eyes containingone or more blebs, and/or by means of a fluid-air exchange. This isparticularly relevant to the central retina since this area generallyresists detachment by subretinal injection.

In some aspects fluid-air exchange is utilized following subretinalinjection; fluid from the infusion cannula is temporarily replaced byair, e.g. from blowing air onto the surface of the retina. As the volumeof the air displaces saline fluid from the vitreous cavity, the bleb iskept in place without efflux into the vitreous cavity. By positioningthe eye globe appropriately, the bleb of subretinal vector in some casescan be manipulated to involve adjacent areas (e.g. the macula and/orfovea). In some cases, the mass of the bleb is sufficient to cause it togravitate, even without use of the fluid-air exchange. Movement of thebleb may be further be facilitated by altering the position of the humansubject's head, so as to allow the bleb to gravitate to the desiredlocation in the eye. Once the desired configuration of the bleb isachieved, fluid is returned to the vitreous cavity. The fluid is anappropriate fluid, e.g., fresh saline. Generally, the subretinal vectormay be left in situ without retinopexy to the retinotomy and withoutintraocular tamponade, and the retina will spontaneously reattach withinabout 48 hours.

Subretinal administration of AAV-2 for treatment of an ocular diseasehas been demonstrated in treatment of the rare genetic disease, Leber'sCongenital Amaurosis (“LCA”). The pathology of LCA and the LCA patientpopulation are different from those of wet-AMD and therefore it was notexpected that treatment of wet AMD with gene therapy, and in particular,with AAV-2, would be safe and effective prior to the rAAV.sFLT clinicalstudy. Specifically, LCA is a degenerative genetic disease caused byinsufficient expression of the retinal protein RPE-65. It causes slowdeterioration of vision in babies and young children that leads to totalblindness by young adulthood, generally prior to age 25 to 30. Bycontrast, as described here previously, wet AMD is caused by growth ofnew blood vessels in the retina late in life, generally beginningbetween age 65-75. The presence of new vessels raises the concern thatAAV particles, the transgene or the transgene product, would betransported outside the eye in greater amounts than was shown in the LCAstudy. Additionally, the immune system and immune response to foreignsubstances changes as patients age creating uncertainly prior to studyresults disclosed in Example 12 that treatment of wet AMD with a viralvector such as rAAV.sFLT-1 would be safe and effective.

B. Effect of Treatment

In some aspects, a single injection of the pharmaceutical composition ofthe present disclosure into the affected eye not only has the benefitsof the Lucentis® treatment, but may also require only one singleinjection.

The pharmaceutical composition of the present disclosure can stopleakage in existing blood vessels and can inhibit further new vesselformation in the subretinal space of patients suffering from CNVsecondary to AMD for at least 18 months, and in some aspects theactivity continues for 3-5 years. Inhibition of leakage and new vesselformation prevents the development of blindness in affected patients.

In some aspects, the sFLT-1 protein levels in the vitreous of said humansubject is about 500-5,000 pg/ml, about 600-4,000 pg/ml, about 800-3,000pg/ml about 900-2,000 pg/ml, or about 1,000-1,800 pg/ml, 500-700 pg/ml,700-1,000 pg/ml, 1,000-1200 pg/ml, 1200-1,500 pg/ml, 1, 800-2000 pg/ml.In some cases, protein levels in the vitreous of the human subject is atleast about 100, 200, 300, 400, 500, 600, 700. 800, 900, 1000, 1100,1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300or 2400 pg/ml. In some cases, protein levels in the vitreous of thehuman subject is at most about 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000,2100, 2200, 2300 or 2400 pg/ml.

In some cases, protein “levels” may refer to any quantity or relativequantity of protein. In some cases, level may be measured as aconcentration (e.g. pM, nM, uM etc.), a molality (e.g. m), as a mass(e.g. pg, ug, ng etc.) or any suitable measurement. In some cases, aunitless measurement may indicate a level.

In some cases, protein levels may be measured at least about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 14, 21 or 30,50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350 or 365days after administering said pharmaceutical composition. In some cases,protein levels may be measured at most about 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 14, 21 or 30, 50, 75, 100,125, 150, 175, 200, 225, 250, 275, 300, 325, 350 or 365 days afteradministering said pharmaceutical composition. In some cases, proteinlevels are measured at least 72 hours after administering saidpharmaceutical composition.

Administration of the pharmaceutical composition of the presentdisclosure general leads to no side effects or adverse events.

In some aspects, no vector is detected in the human subject's tear,blood, saliva or urine samples 7, 14, 21 or 30 clays after administeringsaid pharmaceutical composition. In some aspects, the presence of theviral vector is detected by qPCR or ELISA as known in the art.

In some cases, no vector is detected in the human subject's Lear, blood,saliva or urine samples at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.70.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 14, 21 or 30, 50, 75, 100, 125, 150,175, 200, 225, 250, 275, 300, 325, 350 or 365 days after administeringsaid pharmaceutical composition. In some cases, no vector is detected inthe human subject's tear, blood, saliva or urine samples at most about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 14,21 or 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350or 365 days after administering said pharmaceutical composition. In somecases, no vector is detected in the human subject's tear, blood, salivaor urine samples are measured at least 72 hours after administering saidpharmaceutical composition.

In some aspects, the human subject shows no clinically significantretinal toxicity as assessed by serial ophthalmic examinations over atleast about a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 month months period.In some aspects, the human subject shows no clinically significantretinal toxicity as assessed by serial ophthalmic examinations over atmost about a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 month months period.

In some aspects, no superficial, anterior segment or vitreousinflammatory signs are present in the human subject over least a twomonths period. In some cases, no superficial, anterior segment orvitreous inflammatory signs are present in the human subject at 1 weekor at 3, 6, 9 or 12 months after administration of the pharmaceuticalcomposition.

In some aspects, no inflammatory signs are seen including a cytotoxic Tcell response within about a 10% of normal range following administeringstep. In some aspects, there is no increase in T-cell response asmeasured by ELISpot. In some aspects, T cells do not express HLA-DR orKi67, and do not develop an activated effector phenotype, as describedin Lai et al. 2011; Gene Therapy, which is herein incorporated byreference in its entirety. In some aspects, no inflammation of thevitreous is observed by biomicroscopy (BE) and indirect opthalmoscopy(JOE) following the administering step. In some aspects, traceinflammation of the vitreous that resolved within 10 days is observed bybiomicroscopy (BE) and indirect opthalmoscopy (JOE) following theadministering step. In some aspects, the human subject does not requirerescue treatment at least 120 days post administration. In some aspects,the human subject does not require rescue treatment for at least 30days, at least 60 days, at least 90 days, at least 120 days at least 180days, at least 270 days or at least 365 days after administration.

As used herein, rescue treatment refers to an administration of a doseof a VEGF inhibitor after the initial administration of thepharmaceutical composition described in the present disclosure. A rescuetreatment is administered to boost the amount of VEGF inhibition in theeye patient in order to arrest or reverse signs and symptoms of diseaseprogression. The decision to administer a rescue treatment may be basedon predetermined diagnostic criteria, as in the clinical study describedin Example 12, or on a physician's clinical judgment that signs ofactive disease are present in a patient.

In some aspects, there is no evidence of visual acuity loss, IOPelevation, retinal detachment, or any intraocular or systemic immuneresponse in said human subject at least 120 days post administration. Insome aspects, there is no evidence of visual acuity loss, IOP elevation,retinal detachment, or any intraocular or systemic immune response insaid human subject at least 30 days, at least 60 days, at least 90 days,at least 120 days at least 180 days, at least 270 days or at least 365days after administration. In some aspects, there is no evidence ofvisual acuity loss, IOP elevation, retinal detachment, or anyintraocular or systemic immune response in said human subject at most 30days, at least 60 days, at least 90 days, at least 120 days at least 180days, at least 270 days or at least 365 days after administration.

In some aspects, a patient's best corrected visual acuity (BCVA)improves by 1, 2 3, 4, 5 or more lines.

In some aspects, a reduction in neovascularization as assessed byFluorscein Angiography (FA) follows the administering step.

In some cases, retinal thickness may be measured to examine the effectsof treatment. In some cases, the central retinal thickness of the humansubject does not increase by more than 50 microns, 100 microns, or 250microns within 12 months following treatment with the pharmaceuticalcomposition of the disclosure. In some cases, the central retinalthickness of the human subject decreases by at least 50 microns, 100microns, 200 microns, 250 microns, 300 microns, 400 microns, 500microns, 600 microns within 3 months, 6 months or 9 months 12 monthsfollowing treatment with the pharmaceutical composition of thedisclosure. The decrease in the central retinal thickness of the humansubject may be measured comparing the central retinal thickness at pointin time to a baseline measurement taken at or within 1, 3, 7 or 10 daysof the administration of the pharmaceutical composition of thedisclosure.

C. Combination Treatment with VEGF Inhibitors

In some aspects, the method further comprises administering to the humansubject a pharmaceutically effective amount of a VEGF inhibitor.

In some aspects, the VEGF inhibitor comprises an antibody against VEGFor a functional fragment thereof. In some aspects, the VEGF inhibitorcomprises ranibizumab. In other aspects the VEGF inhibitor is a solublereceptor, fusion protein, or fragment thereof, such as aflibercept orsFLT01. In some aspects, the pharmaceutical composition is administeredat least 1, 2, 3, 4, 5, 6, 7, or 8 days after the administering of saidVEGF inhibitor. In some aspects, the pharmaceutical composition isadministered at most 1, 2, 3, 4, 5, 6, 7, or 8 days after theadministering of said VEGF inhibitor. In some aspects, thepharmaceutical composition is administered within 90 days after theadministering of said VEGF inhibitor.

In some aspects, the patient is treated under a protocol such asoutlined in FIG. 13. After the protein expressed by the recombinantvirus is expressed at a suitable level, (or “on”), the patients arefollowed with criteria-based re-treatment:

-   -   If disease recurs, ranibizumab re-treatment is allowed    -   Expect 5-8 re-treatments per year with control group    -   In treatment group, expect equivalent vision with substantial        decrease in number of re-treatments.

The patient is eligible for re-treatment if signs of active CNV arepresent:

-   -   Based upon objective criteria as evaluated by masked personnel        (technician and ophthalmologist)    -   Re-treatment criteria are based upon substantial experience with        “as needed” (PRN) treatment in previous trials with anti-VEGF        agents.

Re-treatment is warranted based on signs of active disease; such as:

-   -   >10 Early Treatment Diabetic Retinopathy Study (ETDRS) letter        loss from human subject's previous visit (attributable to        retinal causes), OR a decrease of >5 ETDRS letters from previous        visit in conjunction with patient perception of functional loss;    -   Any increased, new, or persistent subsensory, sub-Retinal        Pigment Epithelial (RPE), or intraretinal fluid on OCT;    -   Signs of increased CNV leakage via FA.

In some aspects, the VEGF inhibitor is administered for at least 1 timeprior to administering the said pharmaceutical composition and anadditional 1 or 2 times at about 30 day intervals following saidadministration to prevent disease progression while protein expressionincrease to suitable levels. In some aspects, the VEGF inhibitor isadministered for at least 2 times prior to administering saidpharmaceutical composition. In some aspects, the VEGF inhibitor isadministered over a period of 6 to 7 weeks following administration ofsaid pharmaceutical composition.

In some aspects, the frequency of administration of VEGF inhibitor isreduced by less than a year or stopped altogether.

In some aspects, the present disclosure is used after 3 or moretreatments of VEGF inhibitors. In some aspects, the present disclosureis used after observation that AMD patients show no improvement in BCVAafter use of other VEGF inhibitors.

D. Other Combination Treatments

In another preferred aspect, treatment of a patient comprisesadministration one or more of the pharmaceutical compositions providedherein, in conjunction with other therapies, for example, chemotherapy,radiation, surgery, anti-inflammatory agents, selected vitamins and thelike. The other agents can be administered, prior to, after orco-administered with the pharmaceutical compositions.

Aspects of the disclosure may be practiced without the theoreticalaspects presented. Moreover, the theoretical aspects are presented withthe understanding that Applicants do not seek to be bound by the theorypresented.

While preferred aspects of the present disclosure have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch aspects are provided by way of example only. Numerous variations,changes, and substitutions will now occur to those skilled in the artwithout departing from the disclosure. It should be understood thatvarious alternatives to the aspects of the disclosure described hereinmay be employed in practicing the disclosure. It is intended that thefollowing claims define the scope of the disclosure and that methods andstructures within the scope of these claims and their equivalents becovered thereby.

The effective dose of the nucleic acid will be a function of theparticular expressed protein, the particular disease to be targeted, thepatient and his or her clinical condition, weight, age, sex, etc.

EXAMPLES

It will be understood by those of skill in the art that numerous andvarious modifications can be made to yield essentially similar resultswithout departing from the spirit of the present disclosure. All of thereferences referred to herein are incorporated by reference in theirentirety for the subject matter discussed. The following examples areincluded for illustrative purposes only and are not intended to limitthe scope of the disclosure.

It must be explained, if not specified, that the percentage of followingexamples are all weight percent content wt %.

Example 1 rAAV.sFlt-1

One example recombinant virus is rAAV.sFlt-1. It encodes a vector and ahuman form of the truncated, soluble VEGF receptor 1 (sFLT-1). Thevector is a recombinant, replicative-deficient adeno-associated viral(rAAV) vector, of serotype 2.

The rAAV.sFlt-1 was manufactured under Good Manufacturing Practices(cGMP). At the manufacture site, the final product was aliquoted intosterile, low-virus-binding microcentrifuge tubes (individually wrapped,low-retention, sterilised flat cap vials) according to the protocolrequirements (i.e. 200 μl of 1×10¹⁰ or 1×10¹¹ viral genomes) and storedat −80° C. to await final product release. Each vial contained enoughvector for use in a single patient (100 μl to be administered).

The recombinant virus, rAAV.sFlt-1, is a recombinant adeno-associatedvirus 2 (rAAV2) vector carrying the soluble VEGFR receptor 1 (VEGFR1) orsFLT-1 driven by the human cytomegalovirus (CMV) promoter. TherAAV.sFlt-1 vector and intact AAV2 genome used as the backbone wasprepared as described in Lai et. al. Gene Therapy 2002 vol. 9 (12)804-813). The rAAV2 vector is devoid of viral coding sequences, i.e.,rep and cap have been replaced with an expression cassette for thetherapeutic gene. The active moiety of rAAV.sFlt-1 is sFlt-1. sFLT-1 isthe soluble truncated form of the vascular endothelial growth factorreceptor 1 (VEGFR1 or Flt-1) which occurs naturally. sFLT-1 is the onlyknown endogenous specific inhibitor of VEGF. sFLT-1 is generated byalternative splicing and it lacks the membrane-proximalimmunoglobulin-like domain, the transmembrane spanning region and theintracellular tyrosine-kinase domain. Hence, it contains only the firstsix extracellular immunoglobulin-like loops followed by 31 unique aminoacid residues. sFLT-1 was first identified in human umbilical veinendothelial cells (HUVEC), but it has since been found to occurnaturally in the placenta and circulating systematically in pregnantwomen. The sFLT-1 used in generating rAAV.sFlt-1 contains an openreading frame encoding only the first six extracellularimmunoglobulin-like domains of the full length membrane-spanning FLT-1,followed by a unique 31-amino acid long C-terminal extension,representing the alternatively splices, secreted soluble FLT-1 isoformdescribed earlier.

While the ITR has been shown to possess mild promoter activity, formaximum levels of transgene expression, the cassette generally includesa promoter/enhancer combination, a small intron sequence, the cDNA ofthe therapeutic gene, and a polyadenylation signal. In rAAV.sFlt-1, thehuman CMV major immediate early gene enhancer/promoter and a chimericintron were placed upstream of the sFLT-1 cDNA. A simian virus 40polyadenylation (SV40 poly A) signal was placed downstream of the sFLT-1cDNA.

Binding of sFLT-1 to VEGF in vitro has been widely demonstrated. Theability of sFLT-1 to inhibit VEGF-driven angiogenesis has attractedconsiderable attention for its potential clinical application, but noevidence of efficacy or suitability in humans was shown prior to theclinical study of rAAV.sFlt-1 described in Example 12. The angiostaticactivity of sFLT-1 results from inhibition of VEGF by two mechanisms: i)sequestration of VEGF, to which it binds with high affinity, and ii)formation of inactive heterodimers with membrane-spanning isoforms ofthe VEGF receptors Flt-1 and KDR/Flk-1.

Nucleotide Sequence and Diagram of Plasmid Vector Used to GeneraterAAV.sFlt-1

rAAV.sFlt-1 was generated by triple transfection of human embryonickidney 293 cells with DNA from the pSSV.CI.hsFlt-1 plasmid vector andhelper plasmids, as is known in the art (Xiao et al., 1998. J Virololgy,72(3): 2224-2232). rAAV.sFlt-1 was purified using a sequential processof nuclei isolation, density gradient centrifugation and heparin sulfateaffinity column chromatography. A diagrammatic representation of thesFLT-1 plasmid vector is given in FIG. 1.

Formulation

rAAV.sFlt-1 was formulated in sterile phosphate buffered saline (pH7) at2 concentrations: 1×1010 vector genome/100 μL (low dose) and 1×1011vector genome/100 μL (high dose) in sterile low-virus-bindingmicrocentrifuge tubes. The formulation is preservative-free and is forone-thaw, single use by subretinal injection only.

rAAV(bv).sFlt-1

A second example recombinant virus is rAAV(bv).sFlt-1. rAAV(bv).sFlt-1is a recombinant, replicative-deficient adeno-associated viral (rAAV)vector, of serotype 2 that is produced using a baculovirus expressionsystem (BEVS) in Sf9 insect cells, and encodes a human form of thetruncated, soluble VEGF receptor 1 (sFLT-1). The vector was producedusing infection in Sf9 cells with two recombinant baculovirsues,Bac-inRep-inCap and Bac-sFlt-1. Bac-sFlt-1 was derived from bacmid DNAthat was generated from transformation of electrocompetent cells with an8.7 kb plasmid, AVA01-pFB-CMV-sFlt, which was cloned from theFrag001m-BHKan and the plasmid backbone V109-pFB-AAV-CMV-SV40pA-Kanusing standard molecular biology techniques, as described in Maniatis etal., and as further described below. Frag001m was formed from thefollowing sequential nucleic acide elements which were chemicallysynthesized by Blue Heron Biotech, LLC (Bothell, Wash.) and cloned intoa BHKan backbone: an ITR (AAV serotype 2), CMV-IE promoter, chimericintron, 5′ untranslated region (UTR), sFlt-1 coding sequence, SV40 polyAregion, ITR (AAV serotype 2). The plasmid V109-pFB-AAV-CMV-SV40pA-Kanwas obtained from Virovek, Inc. (Hayward, Calif.). The plasmid containeda kanamycin antibiotic resistance gene, a ColE1 origin and a recombinantAAV cassette, which contained a CMV-IE promoter, an intron, multiplecloning sequences and a SV40 polyA region, flanked by inverted terminalrepeats (ITRs) from AAV serotype 2. This rAAV cassette was flanked by agentamicin resistance gene and Tn7L attachment sites. AVA01-pFB-CMV-sFltdid not contain a T7 RNA polymerase promoter or other prokaryoticregulatory sequence. Bac-inRep-inCap is a recombinant baculoviruscontaining expression cassettes for rep and cap genes from AAV serotype2.

rAAV(bv).sFlt-1 Production in Baculovirus

rAAV(bv).sFlt-1 was produced in baculovirus according to the methodsdescribed in U.S. patent application Ser. No. 12/297,958 and morespecifically as follows: Sf9 cells were grown at 28° C. to about 107cells/ml in SF900 II SFM media containing 100 units/ml of penicillin and100 μg/ml streptomycin, and diluted to about 5×106 cells/ml prior toinfection. Bac-inRep-inCap and Bac-sFlt-1, each at m.o.i. of one wereused to infect the cells at 28° C. for 3 days to produce AAV type 2vectors. After 3 days of infection, cell pellets were collected bycentrifugation at 2,000 rpm for 15 min in a tabletop centrifuge. Thecell pellets were lysed in lysis buffer as described by Urabe et al.,Hum Gene Ther. 1; 13(16):1935-43 (2002) and cellular nucleic acids (DNAand RNA) were digested by benzonase (Sigma, St. Louis, Mo.). The celllysates were cleared by centrifugation at 8,000 rpm for 30 min in anAvanti J-25 centrifuge (Beckman, Fullerton, Calif.) and then loaded ontoan SW28 centrifuge tube containing 5 ml of 1.55 g/cc, and 10 ml of 1.32g/cc of CsCl solutions. After centrifugation at 28,000 rpm for about 16hours at 15° C., the rAAV-containing fraction was collected bypuncturing the centrifuge tube using a syringe needle and subjected to asecond round of CsCl ultracentrifugation. The rAAV-containing fractionwas collected again by puncturing the centrifuge tube using a syringeneedle and dialyzed in PBS buffer to remove the salts and detergents.Vector titers were determined by quantitative real-time PCR assayaccording to manufacturer's protocol (Applied Biosystems, Foster City,Calif.).s

Example 2 In Vitro Inhibition of VEGF-Induced Endothelial CellProliferation

Studies were performed to assess VEGF-induction of human umbilical veinendothelial cell (HUVEC) proliferation and to determine whetherVEGF-induced HUVEC proliferation would be inhibited by rAAV-mediatedsFLT-1. The presence of sFLT-1 in transduced cells was first confirmedby Western blot analysis of conditioned media (FIG. 2, panel a).Conditioned medium from rAAV.sFlt-1-transduced and rAAV.gfp-transduced293 cells were added to VEGF-treated HUVECs in increasing dilutions. Acontrol starvation medium (normal HUVEC growth medium without bovineendothelial growth factor) only was also included. Heparin was added toeach well at 100 μg/mL. The relative VEGF-induced proliferation ofHUVECS treated with VEGF and the different conditioned media was assayedby addition 25 μL of 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrozoliumbromide (MTT, 5 mg/mL, Sigma) to each well for 4 hours at 37° C. Thesecreted sFLT-1 encoded by the rAAV vector in 40 μL of conditionedmedium from rAAV.sFlt-1-transduced 293 cells was confirmed to inhibitVEGF-induced proliferation of HUVECS by 32%. Doubling the volume ofconditioned medium resulted in complete inhibition with cell growthequivalent to basal levels similar to culture in starvation medium (FIG.2, panel b).

In Vitro Assessment of rAAV.sFt-1 Vector Potency

Studies were performed to assess the potency of AAV vectors encoding therecombinant human sFlt-1 gene by quantifying human sFlt-1 proteinexpression of transduced human embryonic kidney 293 (HEK293) cells byELISA. Human embryonic kidney 293 cells were obtained from the AmericanType Culture Collection (Rockville, Md., USA) and cultured in Dulbecco'sModified Eagle Medium (DMEM; Gibco, Grand Island, N.Y., USA) with 10%Fetal bovine serum (FBS, GIBCO) and 1×Penicillin-Streptomycin-Glutamine. All cultures were maintained at 37°C. and 5% CO₂ in a humidified atmosphere.

The HEK293 cells were seeded at 8E4 or 1.5E5 cells/24 well andtransduced at 60-90% confluency with the recombinant AAV vectors at amultiplicity of infection (MOI) ranging from 1×10³-1×10⁶ in DMEM mediumsupplemented with 2% FBS. After 72 hours, post-transduction, conditionedmedia were collected. Aliquots of the conditioned media were preparedfor ELISA using reagents and according to standard instructions from theR&D Systems SVR100B Quantikine ELISA Human sVEGF R1/sFlt-1 kit. (R&DSystems, Minneapolis, Minn.). Samples, standards and controls wereprepared according to the ELISA kit instructions with the R&D SystemsELISA reagents and then transferred to an ELISA plate pre-coated with anantibody to sVEGF R1/sFlt-1 and incubated for two hours at roomtemperature on a horizontal orbital microplate shaker. After incubation,anti-sVEGF R1 Conjugate (two hours), substrate solution (30 minutes) andstop solution were sequentially applied to each well with aspiration andwash steps between each according to standard ELISA assay procedures.The optical density (OD) of the samples, standards and controls wasmeasured within 30 minutes of stopping the substrate reaction with anELISA plate reader. The concentration of sFlt-1 in pg/mL was calculatedusing SoftmaxPro software using the OD measurements from the ELISA platereader.

Results of the studies for rAAV.sFlt-1 and rAAV(bv).sFlt-1 are presentedin FIG. 25A and FIG. 25B. The concentration of sFlt-1 protein expressedby HEK293 cells 72 hours after transduction with rAAV.sFlt-1 andrAAV(bv).sFlt-1 ranged from 100-1,000 pg/mL at an MOI of 1×10⁴,100-10,000 pg/mL at an MOI of 1×10⁵ and 1,000-10,000 pg/mL at an MOI of1×10⁶.

Example 3 rAAV.sFlt-1 Studies in Mice

Transgenic mice (trVEGF029) with slow, but stable retinalneovascularization induced by transgenic expression of human VEGF fromphotoreceptor cells were used as a model for retinal neovascularization.Two separate studies with these mice have been conducted.

In the first mouse study, 13 transgenic mice were assessed for ocularneovascular changes before and after administration of the rAAV.sFlt-1vector (1×10¹¹ vector particles) in one eye and control vector in thecontralateral eye. Eyes were assessed for neovascular changes usingfluorescein angiography at one, three and eight months after injection.The extent, intensity and stage of neovascularization were graded (0-4)by three observers, masked to the treatment received in the eyesexamined. There was a statistically significant overall reduction in theneovascular grading from a median grade of ‘3’ (before injection) to amedian grade of ‘1’ at one month after injection (P=0.012). Thisreduction was maintained at three months (median=1; P=0.001) and ateight months (median=1; P=0.001) after injection with rAAV.sFlt-1.Injection of rAAV.sFlt-1 vector resulted in the long-term (at leasteight months) regression of neovascular vessels in 85% (11 of 13) oftreated eyes compared to 8% (1 of 13) in the control vector-treatedeyes.

Histological examination of the eyes in this preclinical study revealedthat disturbance or loss of photoreceptors was significantly (P<0.01)more pronounced in control vector-injected eyes compared to eyesinjected with rAAV.sFlt-1. Expression of sFLT-1 was also confirmed byreverse transcriptase-polymerase chain reaction analysis of tissuesamples; mRNA for sFLT-1 was detected in all four eyes tested. NorAAV.sFlt-1 vector-specific adverse effects were noted in the eyeinjected with rAAV.sFlt-1 when compared to the eye injected with thecontrol (rAAV.gfp) vector.

In the second study, conducted in trVEGF02 transgenic mice, the aim ofthe study was to determine whether subretinal injection of rAAV.sFlt-1resulted in any cell-mediated immune responses that could negativelyimpact on long-term expression of sFLT-1 or cause immuneresponse-associated damage to the retina. In this study, 50 trVEGF02transgenic mice were given subretinal injections of rAAV.sFlt-1 (8×109viral particles) or phosphate-buffered saline (PBS) in one eye. Theretinas of 30 mice from either the rAAV.sFlt-1 or control treatmentgroups were then assessed at one week and one month post-injection forthe presence of immune cells (leucocytes, macrophages and B- andT-lymphocytes). Flow cytometric examination of the posterior eye cupshowed that at one week post-injection there was a statisticallysignificant increase in CD45+ leucocytes (6.6-fold increase compared tocontrol; P<0.05) and CD11b+ macrophages (5.7-fold increase compared tocontrol; P<0.036). However, there were no differences in CD19+, CD8+ andCD4+ (B- and T-lymphocytes) at this time point. At one monthpost-injection, there were no differences in cell numbers betweenleucocyte subsets (i.e. CD45+, CD19+, CD11b+, CD8+ or CD4+ cells) in themouse eyes treated with rAAV.sFlt-1 or the PBS control, suggesting thatthe infiltration of leucocytes and macrophages was transient. Flowcytometric evaluation of lymphocyte subsets of the spleens from thesemice at the one-week and one-month time points showed no significantdifferences in the numbers of lymphocytes. This finding suggests thatthere was no systemic immune response observed, albeit a transient,localized immune response had been shown in the retina.

In this second study, histological examination of the eyes from five ofthe mice injected with either rAAV.sFlt-1 or PBS revealed no observableimmune-response associated destruction or sequelae in the retinas of anyof the mice examined.

To assess the impact of rAAV.sFlt-1 on the level of neovascularizationin this transgenic mouse model (trVEGF02) of retinal neovascularization,the retinas of the mice injected with either rAAV.sFlt-1 or PBS werealso graded independently by two different assessors at two months aftertreatment. Overall, there was a significant reduction in meanneovascularization grades (before injection: 1.46±0.58; after injection:0.81±0.57; P<0.00015) in the rAAV.sFlt-1-injected eyes whereas there wasa significant increase in mean neovascularization grades (beforeinjection: 1.08±0.56; after injection: 1.63±0.96; P<0.018) in the PBScontrol-injected eyes.

The findings from this second mouse study clearly indicate thattreatment with rAAV.sFlt-1 appeared to reverse the progressive increasein neovascularization observed in this mouse model of retinalneovascularization and AMD. Furthermore, only a limited, localized,inflammatory response was observed one week after subretinal injectionwith rAAV.sFlt-1 and resolved at one month. This immune response did notappear to compromise the long-term therapeutic efficacy of rAAV.sFlt-1in the retina.

The transgenic mice models described in this Example 3 demonstrate thatthe pharmaceutical compositions disclosed herein can be used for thetreatment and/or prophylaxis of other retinal vascular diseases in whichVEGF inhibition is implicated. These include diabetic macular edema,diabetic retina ischemia, diabetic retinal edema, proliferative diabeticretinopathy, retinal vein occlusion, central retinal vein occlusion andbranched retinal vein occlusion. In clinical studies some VEGFinhibitors, such as Lucentis, have been shown to effectively treatcertain of these diseases including diabetic macular edema and retinalvein occlusion. The efficacy of rAAV.sFlt-1 demonstrated in these mousemodels indicates rAAV.sFlt-1 is also effective in treating these VEGFmediated diseases.

Example 4 rAAV.sFlt-1 Study in Rats

In the rat rAAV.sFlt-1 study, two models of ocular neovascularizationwere used: cautery-induced corneal neovascularization and laserphotocoagulation-induced choroidal neovascularization (CNV). In thecorneal neovascularization model, 22 rats were injected with rAAV.sFlt-1vector (8×10⁸ viral particles) in the anterior chamber of one eye andwith control vector (rAAV.gfp) in the contralateral eye, followed bycauterization of the cornea. The eyes were then examined forneovascularization four days after cautery, using slit-lamp photography.A significantly lower rate of corneal vascularization was found in therAAV.sFlt-1-treated eyes compared to the control-treated eyes (27% and63%, respectively; P=0.009). Histological examination of the eyes showedthat no corneal blood vessels were observed in the majority ofcauterized, rAAV.sFlt-1-treated eyes. Histological examination alsorevealed that cellular infiltration of the corneal stromal layer wasmore pronounced in the control vector-injected eyes compared to therAAV.sFlt-1-treated eyes. In addition, there was obvious edema andcorneal stroma swelling in the control vector-treated eyes whereas therewas no evidence of significant tissue swelling in rAAV.sFlt-1-treatedeyes.

In the laser photocoagulation-induced CNV model, 10 rats were injectedsubretinally with rAAV.sFlt-1 vector (8×108 viral particles) in one eye,and a control vector (rAAV.gfp) in the contralateral eye. Laserphotocoagulation was used to induce CNV one month after injection. Fiveweeks after laser photocoagulation, eyes were examined for CNV usingfluorescein angiography. Only 41% of the laser-treated areas showedleakage in the rAAV.sFlt-1 treated eyes compared to 60% in the controlvector-treated eyes (P=0.002). Sixteen weeks after laser-induced CNV,the rAAV.sFlt-1-treated eyes still showed significantly lowerneovascularization than control eyes. Histological examination of theeyes in the areas immediately adjacent to the injection sites revealed anormal retinal pigmented epithelium and normal outer segments and outernuclear layer. These findings suggested there was no obvious toxicityassociated with sFLT-1 expression. Electroretinograms also indicatednormal functioning of rAAV.sFlt-1-treated eyes. Most of the rAAV.sFlt-1and control vector-treated laser lesions developed subretinal cellularmembranes. However, the lesions in eyes treated with rAAV.sFlt-1generally had less proliferating endothelial cells, reflecting thefluorescein angiography findings, and indicating that the rate ofangiogenesis (i.e. neovascularization) was reduced inrAAV.sFlt-1-treated eyes.

rAAV.sFlt-1 and rAAV(bv).sFlt-1 Study in Rat Model of Diabetes

To further assess the safety and efficacy of rAAV.sFlt-1 andrAAV(bv).sFlt-1 for the treatment of diabetic retinopathy (DR) anddiabetic macular edema (DME), an experiment in a rat model of diabetesis conducted.

Vision loss in diabetic patients is mediated by inflammation, leading tothe eventual breakdown of the blood-retinal-barrier and subsequentvascular leakage, resulting in macular edema. The streptozotocin(STZ)-diabetic rat model displays a well-characterized pattern ofvascular leakage, in which VEGF is strongly upregulated as early as 2weeks. (Miyamoto, K., et al. Proc Natl Acad Sci USA 96, 10836-10841(1999). Current approaches to treating animal models of DR demonstrateonly a partial resolution of vascular leakage.

Diabetes is induced in Brown Norway rats by intraperitoneal injection ofstreptozotocin (50 mg/kg). Diabetes is confirmed and monitored by bloodglucose measurements. Rats with blood glucose >350 mg/dl are considereddiabetic. Eight days following onset of diabetes, rats are treated bysubretinal injection (n=12 eyes per group) with 5 μL containing either1×10¹⁰ or 5×10¹⁰ vg of rAAV.sFlt-1 or rAAV(bv).sFlt-1 using establishedtechniques as described in Chalberg, T. W. et al., Invest Ophthalmol VisSci 46, 2140-2146 (2005). AAV2.GFP (5×10¹⁰ vg) and vehicle are beinjected as controls. Non-diabetic and diabetic no-treatment groups arealso used as controls.

The effect of the rAAV(bv).sFlt-1 expressing sFLT-1 on vascular leakageis measure at 60 days. Retinal vascular leakage is measured by theFITC-albumin leakage method following the injection using theFITC-conjugated albumin as tracer. The FITC-albumin leakage methoddirectly measures the leakage of FITC-albumin leaking into the retinafrom the circulation and is a commonly used method to measure retinalvascular permeability. Retinal vascular leakage in injected eyes will becompared to non-diabetic controls, untreated and vehicle-treateddiabetic eyes, and wildtype AAV serotypes 2 and 8.

Results: rAAV(bv).sFlt-1 expressing sFLT-1 reduces vascular leakage inthe STZ-diabetic rat whereas injection of AAV2.GFP and other controlsdoes not.

Example 5 rAAV.sFlt-1 Study in Monkeys

The efficacy and safety of rAAV.sFlt-1 was also examined in a nonhumanprimate (macaque) model of AMD using laser photocoagulation to induceCNV. One challenge in developing treatments for AMD in humans is thatnonhuman primates do not develop AMD. Laser photocoagulation induced CNVsimulates some symptoms of AMD, but the underlying biological process ishealing of an acute injury rather than progression of a chronic diseaseand thus may not be predictive of the performance of any particulartreatment for CNV in humans with AMD or other CNV based diseases.Nonetheless, because human eyes are anatomically more similar tononhuman primate eyes than nonprimate eyes, nonhuman primates arefrequently studied to assess toxicity and histological response to apotential treatment or other intervention.

In the first study on nonhuman primates, five macaque monkeys wereinjected subretinally with rAAV.sFlt-1 (4×10¹² viral particles) in oneeye, and a control vector (rAAV.gfp) in the contralateral eye. The eyehealth of the monkeys was periodically assessed after subretinalinjection. There was no apparent complication related directly tosubretinal injection of either the control or rAAVsFlt-1 vector. Atransient conjunctival irritation and vitreous haze was noted in theweek following injection, which cleared by the second week. Subretinalinjection was unsuccessful in the right eye of one of the monkeys;therefore this animal was not subjected to further evaluation.

Subretinal injection of 40-100 μL of rAAV suspension lifted the retina,creating a bleb that housed the vector between the pigment epitheliumand the photoreceptor layer in a localized manner. This blebself-corrected within 24 to 48 hours. Except for a minor disturbance tothe retinal pigment epithelium at the point of needle penetration, noother retinal abnormalities were observed for the duration of thefollow-up (3 to 17 months post-injection). No other abnormalities oradverse events were observed; at no time was retinal detachmentassociated with the surgery.

To assess the long-term therapeutic efficacy of rAAVsFlt-1, the fourinjected monkeys were then subjected to intense laser photocoagulation16 months after treatment with the vectors. Eight lesions were inducedusing laser in each eye, and the eyes then monitored for CNV at two andfour weeks after laser treatment. After laser photocoagulation, onlythree of the four monkeys were analyzable, therefore, efficacy data wascollected for three animals. None of three monkey eyes treated withrAAVsFlt-1 developed CNV-related lesions and only weak fluoresceinstaining was observed, indicating minimum leakage/neovascularization.All contralateral eyes treated with control vector developed CNV-relatedlesions.

In a follow-up study aimed at assessing the safety and toxicity ofrAAV.sFlt-1 injected into the subretinal space, eight monkeys were used:five were injected in their left eyes with rAAV.sFlt-1, two injected intheir left eyes with rAAV.gfp, one injected in both eyes withrecombinant Flt-1 protein and one was kept as uninjected control. Themonkeys were examined preinjection and post injection by color fundusphotography, fluorescein angiography and electroretinography. Blood wascollected routinely for assaying sFLT-1 levels and peripheral bloodlymphocytes were isolated for flow cytometry to assess immune cellsubset response. At time of sacrifice (3, 9 and 12 months postinjection), tissues were collected for i) biodistribution studies on therAAV.sFlt-1 vector using real-time polymerase chain reaction onextracted genomic DNA; ii) hsFlt-1 protein and AAV2 capsid protein levelquantitation by ELISA; and iii) histology of the eyes.

Color fundus photography, fluorescein angiography andelectroretinography did not detect any adverse effect on the eyefollowing injection. Plasma sFLT-1 level did not show any rAAV.sFlt-1injection-related rise in level in any of the male or female monkeysexamines. Except for an optic nerve sample, the rAAV.sFlt-1 sequence wasnot detected in the genomic DNA of any of the other tissues sampled(lymph nodes, spleen, liver, brain, brain, heart, spleen, cornea).Haematoxylin and eosin stained paraffin-embedded sections of the eyesappeared normal.

While non-human primate anatomy is more similar to human anatomy thanthe anatomy of smaller mammals such as mice, limitations do exist whichmake studies in non-human primates intriguing, but not predictive ofclinical results in humans. As noted above, the study in this exampleuses a laser injury model in which the animal has otherwise healthyretinal tissue. The retinal tissue was not degraded over time as indisease retinal tissue nor are the disease specific pathogenic factorspresent. Non-human primates frequently differ from humans with respectto biodistribution, pharmacokinetics and dose dependencies, antibodytiter, immune response and inflammatory response in ways that are notpredictable. Additional differences include the ILM (inner limitingmembrane) and the volume of the vitreous chamber, which is approximatelyfour times larger in humans than the nonhuman primates used in thisstudy. The human inner limiting membrane, a barrier that acts to limittransport between the retina and the vitreous, is a more a more profoundand effective barrier than the ILM of a monkey.

Example 6 Safety Studies

In these studies, sFLT-1 protein was measured in the vitreous and plasmaof animals using an enzyme linked immunosorbent assay kit for sFLT-1protein detection. sFLT-1 protein level was upregulated in vitreous andeyes of animals injected with rAAV.sFlt-1. FIG. 3A shows the vitreoussFLT-1 protein level in monkey eyes injected with rAAV.sFlt-1 (left eye)and control eye injected with rAAV.gfp and uninfected eyes (right eye).sFLT-1 protein levels were significantly higher in four out of the fiverAAV.sFlt-1 injected eyes. Table 5.3.1 shows the sFLT-1 protein level inthe mouse eyes that were not injected and that were injected withrAAV.sFlt-1 and enucleated at one month post injection. Overexpressionof sFLT-1 in the eyes of mice and vitreous of monkeys did not have anyadverse effect on their overall well-being. In monkeys, sFLT-1overexpression in the vitreous did not have any effect on their retinalfunction and did not have any clinically or histologically evident toxiceffects on the eyes. The significantly higher sFLT-1 protein levels inthe rAAV.sFlt-1 injected eyes suggests long-term rAAV-mediated hsFLT-1expression and supports previous data on detection of viral mRNAsequence and presence of rAAV-mediated gfp expression in monkey retina17 months post injection.

jh

TABLE 1 Summarizing hsFLT-1 protein levels in rAAV.sFlt-1-injected mouseeyes and uninjected mouse eyes at 1 month post injection. Animal Timepost sFLT-1 species and injection protein level number of eyes Treatment(week) (pg/mL) Mouse (n = 1) uninjected NA 101.4 ± 4.8 Mouse (n = 1)uninjected NA  91.0 ± 10.9 Mouse (n = 1) uninjected NA 113.4 ± 6.3 Mouse(n = 1) uninjected NA 160.2 ± 8.9 Mouse (n = 1) rAAV.sFlt-1 injected 41034.7 ± 44.3 Mouse (n = 1) rAAV.sFlt-1 injected 4  610.3 ± 16.3 Mouse(n = 1) rAAV.sFlt-1 injected 4 1417.2 ± 50   Mouse (n = 1) rAAV.sFlt-1injected 4 >max

Plasma hsFLT-1 levels in the monkeys did not show any trend at thedifferent sampling times (FIG. 3B). This suggests that the injection ofrAAV.sFlt-1 did not have an obvious effect on the plasma hsFLT-1 level.The fluctuating levels did not have any effect on the well-being of themonkeys.

TABLE 2 Immunogenicity Studies Species/ Method of Duration of GLP Strainadministration dosing Doses compliance C57Bl/ subretinal 1, 2 and 8 ×10⁹ vector No 6 mice 4 weeks genomes Monkeys subretinal 12 months 8 ×10¹¹ vector No genomes Table 2: Summary of animal strain, injectionroute, duration and dose of rAAV.sFlt-1 used in immunogenicity studies

Example 7 Immunogenicity Studies on Mice

The cellular immune response to rAAV.sFlt-1 therapy was assessed in themouse eye one, two and four weeks post injection using flow cytometry.Infiltrating leucocytes were identified on the basis of CD45 expressionand classified as monocytes/granulocytes, B cells, CD4⁺ T cells and CD8⁺T cells on the basis of CD11b, CD19, CD4 and CD8 expression,respectively. The posterior eye cup was collected from five mice in eachgroup (rAAV.sFlt-1-injected, PBS-injected, uninjected control) andpooled for analysis. As shown in FIG. 4A, there was no difference in thenumber of cells recovered from each group of mice over the course ofthis experiment. However, there was a significant increase in the numberof CD45⁺ cells one and two weeks post injection that disappeared by fourweeks (FIG. 4B). Almost all of the increase seen at one week could beattributed to an increase in CD11b⁺ cells (FIG. 4C), since there was nodifference in the number of CD4⁺, CD8⁺, and CD19⁺ cells (FIGS. 4D-F). Attwo weeks though, there was no longer a significant difference in thenumber of CD11b⁺ present in the eyes of AAV.sFlt-1 injected mice;instead, there was a significant increase in the number of CD4⁺ and CD8⁺T cells and a possible trend towards an increase in B cells. The numberof CD4⁺ and CD8⁺ cells fell sharply at four weeks yet remainedsignificantly increased compared to the PBS-injected and uninjectedmice. In contrast, there was no change in the number of CD11b⁺, CD4⁺,CD8⁺ and CD19⁺ cells in the spleen during the course of this experiment(FIGS. 5A-E).

The function of the T cells infiltrating the retina was examined moreclosely by stimulating them with PMA/ionomycin or anti-CD3 and measuringintracellular IFN-γ production by flow cytometry.

FIG. 6 shows that compared to uninjected controls, a small proportion ofboth CD4⁺ and CD8⁺ T cells were primed to produce IFN-γ after theinjection of rAAV.sFlt-1. The frequency of IFN-γ producing cells did notvary significantly over the course of the experiment despite an apparentincrease amongst CD8⁺ T cells on day 3 (FIG. 6B). Lower levels of IFN-γwere measured when the T cells were restimulated with a class IMHC-restricted epitope of rAAV capsid protein and some IFN-γ was alsodetected in the absence of any stimulation (data not shown). Takentogether, these results indicate a small proportion of the T cellsinfiltrating the eyes of rAAV.sFlt-1-injected mice had been recentlyactivated to produce IFN-γ, but this did not vary amongst either T cellsubset during the course of this experiment.

The data presented for these experiments on the infiltration of immunecells into the eyes of AAV-sFLT-1 injected mice clearly show two wavesof cell infiltration. There was an early wave of CD11b⁺ cells at 1 weekfollowed by a wave of CD4⁺ and CD8⁺ T cells at 2 weeks. Importantly,neither wave of infiltration was still present at 4 weeks, suggestingthe infiltration had resolved itself. Importantly, sFLT-1 proteinproduction was did not wane at this point, and indeed, continued to beexpressed at very high levels.

The data on IFN-γ production indicated that around 5% of the CD4⁺ andCD8⁺ T were recently primed, and this frequency did not vary over thecourse of the experiment. Hu. et al first described the breakdown of theblood-retinal barrier by activated T cells, and the data presented hereis consistent with the infiltration of activated CD4⁺ and CD8⁺ cells.However, there was no evidence of an increase in the number ofcapsid-specific T cells amongst this population since restimulation withspecific peptide only revealed low and levels of IFN-γ production thatdid not change over the course of the experiment. Taken together, theseobservations suggest that the initial insult that occurred withinjection of rAAV.sFlt-1 produced a short-lived wave of immune cellinfiltration that resolved itself within four weeks, but failed toelicit an ongoing immune response that could harm the tissues of the eyeor affect sFLT-1 expression.

Example 8 Immunogenicity Studies on Monkeys

Immune response following subretinal injection of rAAV.sFlt-1 orrAAV.gfp was analyzed using a panel of antibodies that would identifychanges in immune cell subset populations. The results are summarized inFIG. 4. In some monkeys, very small changes in immune cell subsetpopulations were observed but they were not statistically significant.Despite this, this was followed by a more in-depth study of circulatingcells. Specifically, we assessed the possibility that either the vector(rAAV) or the inserted gene product (sFLT-1) may cause immuneactivation. Activation of B cells and T cells was investigated (FIG. 5and FIG. 6). Other lymphocyte populations were also analyzed todetermine whether the therapy caused any observable differences that maybe indicative of direct activation or a response to activation. Analysiswas conducted using a combination of classic markers (Pitcher, 2002#129), as well as a novel phenotypic analysis described in a recentlypublished report (Miller, 2008 #126). Using a small subset of phenotypicmarkers (HLA-DR, Ki-67, and Bcl-2) we investigated whether followingadministration of rAAV-sFLT-1 CD4+ or CD8+ T cells and/or B cells showedsigns of activation. In the studies published by Miller and colleagues,activated T cells display an activated effector phenotype characterizedby the expression of the differentiation marker HLA-DR and the cellcycle associated nuclear antigen Ki-67, which is used as a marker forproliferation. Resting T cells do not express Ki-67, whereas cycling orrecently divided T cells upregulate Ki-67 expression. A level of Ki-67expression is normally detected as part of homeostatic cell cycling.

Example 9 Biodistribution of rAAV.sFlt-1

Genomic DNA was extracted from tissues collected (optic nerve, lymphnode, brain, heart, lungs, spleen, liver, cornea) immediately aftereuthanasia of monkeys. Real time polymerase chain reaction was performedon the genomic DNA to determine whether the rAAV.sFlt-1 vector constructinjected in the subretinal space would be present elsewhere. Based oncomparison of Ct values between known amounts of control plasmidpssv.C1.sflt-1 DNA, the rAAV.sFlt-1 construct was found at low gene copynumber in the optic nerve of one injected eye and not in any of theother tissues samples. This suggests that rAAV.sFlt-1 injected into thesubretinal space remains mainly within the eye. Table 4 is a summary ofthe Ct values from genomic DNA extracted from monkeys that were notinjected or injected with rAAV.sFlt-1- and rAAV.gfp.

TABLE 3 Ct values and Ct standard deviation values for the differentgenomic DNA and control plasmid DNA samples analyzed. Sample ID Ct MeanCt Std Dev No DNA 0 copy 40.83970125 0.08415232 pssv.C1.hsFlt-1 (0.045ng) 6000000 copies 18.17500393 0.522299978 pssv.C1.hsFLT-1 (0.009.ng)1000000 copies 22.5311632 0.318372962 pssv.C1.hsFLT-1 (0.0009 ng) 100000copies 26.23701276 0.183232131 pssv.C1.hsFLT-1 (0.00009 ng) 10000 copies25.2483849 0.164140658 pssv.C1.hsFLT-1 (0.000009 ng) 1000copies29.4265616 0.415926721 Control uninjected monkey 1 LE Optic nerve 42.110.573 2 RE Optic nerve 43.58 0.323 3 axillary LN 45.86 1.319 4 cervicalLN N/A N/A 5 spleen 40.71 0.093 6 liver 44.16 0.604 Monkey 999:rAAV.sFlt-1 injected, euthanized 3 mo p.i. 7 LE optic nerve 39.13 0.1378 RE optic nerve 42.25 0.153 9 axillary LN 40.87 0.728 10 submandibularLN 40.54 0.453 11 spleen N/A N/A 12 liver 41.23 0.388 Monkey 8294:sFLT-1 protein injected, euthanized 3 mo p.i. 13 LE optic nerve 42.150.545 14 RE optic nerve 42.67 0.411 15 axillary LN 43.92 0.304 16submandibular LN N/A N/A 17 spleen N/A N/A 18 liver 40.45 0.981 Monkey8524: rAAV.sFlt-1 injected, euthanized 9 mo p.i. 19 left cornea 39.720.975 20 right cornea N/A N/A 21 axillary LN 44.12 0.216 22 cervical LNN/A N/A 23 spleen 37.91 0.668 24 liver 41.8 0.648 Monkey 8514:rAAV.sFlt-1 injected, euthanized 12 mo p.i. 25 right optic nerve 39.960.609 26 left optic nerve 28.9 0.057 27 axillary LN 40.08 0.221 28cervical LN 41.27 0.063 29 spleen 39.22 0.196 30 liver 40.79 0.367 31brain 41.14 0.798 32 heart 42.19 0.265 33 lungs 40.11 2.093 Monkey 8523:rAAV.sFlt-1 injected, euthanized 12 mo p.i. 34 left optic nerve 37.270.838 35 right optic nerve 37.92 1.181 36 axillary LN 38.55 0.895 37cervical LN 39.68 0.583 39 spleen 36.44 0.519 40 liver 39.94 0.768 418523 brain 40.29 0.397 42 8523 heart 41.28 0.877 43 8523 lungs 41.711.186 Monkey 8530: rAAV.sFlt-1 injected, euthanized 12 mo p.i. 44 leftoptic nerve 38.52 0.777 45 right optic nerve 40.67 1.354 46 axillary LN42.49 0.841 47 cervical LN 38.55 0.895 48 spleen 36.44 0.519 49 liver39.94 0.768 50 brain 40.29 0.397 51 heart 41.67 1.787 52 lungs 39.291.474 Monkey 8532: rAAV.sFlt-1 injected, euthanized 12 mo p.i. 53 leftoptic nerve 35.07 1.06 54 right optic nerve 38.14 0.665 55 axillary LN40.23 1.171 56 cervical LN 40.82 0.496 57 spleen 40.09 0.195 58 liver40.63 1.1052 59 brain 38.68 0.295 60 heart 40.04 0.685 Monkey 8297:rAAV.sFlt-1 injected, euthanized 12 mo p.i. 61 left optic nerve 39.841.034 62 right optic nerve 42.17 1.247 63 axillary LN 41.19 2.174 64cervical LN 41.38 2.040 65 spleen 39.09 1.273 66 liver 41.36 0.683 67brain 37.84 1.243 68 heart 40.74 0.868 69 lungs 42.60 0.276

Example 10 Efficacy Studies on a Mouse Model of RetinalNeovascularization

Transgenic mice generated through VEGF upregulation in thephotoreceptors cells were used in the study. One eye was injected withrAAV.sFlt-1 and the contralateral eye was injected with rAAV.gfp. Theextent, intensity, and stage of neovascularization were graded by maskedobservers based on an agreed scale. The results shown that there was astatistically significant overall reduction in neovascularization gradesfrom a median of 3 (severe) to a median of 1 (mild) at one month postinjection (P=0.012). This low level of fluorescein leakage wasmaintained at three (median=1; P=0.001) and eight months (median 1;P=0.001) post-rAAV.sFlt-1 injection suggesting the long-term, sustainedtherapeutic effect of rAAV.sFlt-1.

TABLE 4 Grading of eyes before and after AAV.sFlt-1 and AAV.gfpinjection and photoreceptor numbers/rows at 8 months post-injectionGrades at time (months) Photo- Rows of Animal post injection receptorphoto- ID ^(a)0 1 3 8 numbers receptors Regression 243 L 1 0 0 0 68.8 ±14.1 ^(b) 4-8 Moderate 243 R 1 3 3 3 0 0 None 244 L 2 1 1 1 72.8 ± 18.8^(b) 3-7 Moderate 244 R 1 3 2 1 5.8 ± 3.1   0-1 None 247 L 2 2 0 0 80.8± 31.0 ^(b) 3-8 Significant 247 R 1 1 1 1 28.3 ± 33.3   0-4 None 249 L 31 1 1 0 0 Significant 249 R 3 3 3 4 7.2 ± 13.1  0-1 None 250 L 3 1 1 165.1 ± 24.4 ^(b) 3-8 Significant 250 R 3 3 3 3 0 0 None 251 L 3 2 1 1 00 Significant 251 R 3 3 3 4 0 0 None 253 L 3 2 1 1  73.8 ± 20.39 ^(b)5-7 Significant 253 R 3 2 3 2  20 ± 30.3 0-2 Moderate 254 L 3 1 0 0 61.8± 14.3 ^(b) 4-6 Significant 254 R 2 2 2 2 8.8 ± 9.6   0-1 None 324 L 1 11 1 ND ND None 324 R 1 1 1 1 ND ND None 326 L 2 1 1 1 ND ND Moderate 326R 2 2 2 2 ND ND None 327 L 2 2 0 0 ND ND Significant 327 R 2 3 2 2 ND NDNone 329 L 3 2 2 2 ND ND Moderate 329 R 2 2 2 2 ND ND None 330 L 3 3 2 3ND ND None 330 R 3 3 3 3 ND ND None L = left eye injected withAAV.sFlt-1, R = right eye injected with AAV.GFP, ND = not done ^(a)3days prior to injection with AAV vectors. ^(b) Statistically significantdifference in photoreceptor numbers (p < 0.01)

Example 11 Efficacy Studies on a Monkey Model of Laser-Induced ChoroidalNeovascularization

Five monkeys were injected in one eye with rAAV.sFlt-1 and in the otherwith rAAV.gfp. Subretinal injection was unsuccessful in the right eye ofone of the monkeys; therefore this animal was not subjected to furtherevaluation. Subretinal injection of 40-100 μl of rAAV suspension liftedthe retina, creating a bleb that housed the vector between the pigmentepithelium and the photoreceptor layer in a localized manner. This blebself-corrected within 24 to 48 hours. Except for a minor disturbance tothe retinal pigment epithelium at the point of needle penetration, noother retinal abnormalities were observed for the duration of thefollow-up (3 to 17 months post-injection). No other abnormalities oradverse events were observed; at no time was retinal detachmentassociated with the surgery.

To assess the long-term therapeutic efficacy of rAAVsFlt-1, the fourinjected monkeys were then subjected to intense laser photocoagulation16 months after treatment with the vectors. Eight lesions were inducedusing laser in each eye, and the eyes then monitored for choroidalneovascularization at two and four weeks after laser treatment. Afterlaser photocoagulation, only three of the four monkeys were analyzable,therefore, efficacy data was collected for three animals. None of thethree monkey eyes treated with rAAVsFlt-1 developed choroidalneovascularization-related lesions and only weak fluorescein stainingwas observed, indicating minimum leakage/neovascularization. Allcontralateral eyes treated with control vector developed choroidalneovascularization-related lesions. Efficacy data for the three animalsare presented in Table 5.

TABLE 5 Effect of subretinal administration of rAAV.sFlt-1 or control(rAAV.gfp) vector on laser-induced CNV in macaque monkeys CNV Lesionsafter Fluorescein Fundus Angiography^(†) Right Eye Left Eye Time oflaser- (rAAV.sFlt-1) (rAAV.gfp) Monkey induced CNV 2 4 2 4 No. (months)*Weeks Weeks Weeks Weeks 1 16 0/8 0/8 1/8 6/8 2 16 0/8 0/8 0/8 3/8 4 160/8 0/8 0/8 2/8 *CNV was induced at 16 months after subretinal injectionof rAAVs. ^(†)Number of macular lesions with neovascularization(fluorescein leakage) after laser photocoagulation. The retinal functionof the monkeys was assessed by electroretinography. Amplitudes andimplicit times from the responses of the injected eye and uninjectedcontralateral eye were calculated and compared pre-injection and atdifferent times following injection. The results showed that injectionof rAAV.sFlt-1, the recombinant sFLT-1 protein or rAAV.gfp did not haveany adverse effect on the retinal function of the monkeys.

Example 12

The standard of care in treating wet AMD involves frequent intraocularinjection of recombinant anti-VEGF proteins every 4-8 weeks. A rAAVconstruct has been developed for a potent (Kd˜10 pM), naturallyoccurring anti-VEGF protein, soluble Fms-related tyrosine kinase-1(sFlt-1), for the treatment of wet AMD. rAAV.sFlt-1 was produced inaccordance with FDA and ICH guidelines at the UNC Vector Core HumanApplication Laboratory. An eight patient controlled study on the safetyand efficacy of rAAV.sFlt-1 was conducted. Eligibility, inclusion andexclusion criteria for the study were as follows:

Eligibility Criteria

Ages Eligible for Study: 65 Years and older

Genders Eligible for Study: Both

Accepts Healthy Volunteers: No

Inclusion Criteria:

-   -   Age greater than or equal to 65 years;    -   Subfoveal CNV secondary to AMD and with best corrected visual        acuity of 20/80-20/400 or better in the other eye;    -   Fluorescein angiogram of the study eye must show evidence of a        leaking subfoveal choroidal neovascular lesion;    -   Must be a candidate for anti-VEGF intravitreal injections;    -   The entire dimension of the lesion must not exceed 12 Macular        Photocoagulation

Study disc areas;

-   -   No previous retinal treatment of photodynamic therapy or laser;    -   Able to provide informed consent;    -   Participant has clinically acceptable laboratory and ECG at the        time of enrolment; and    -   Able to comply with protocol requirements, including follow-up        visits.

Exclusion Criteria:

-   -   Liver enzymes >2× upper limit of normal;    -   Clinical evidence of active infection of any type, including        adenovirus, hepatitis A, B, or C, or HIV virus;    -   Any prior treatment for AMD in the study/control eye, excluding        anti-VEGF injections;    -   A tear in the retinal pigmented epithelium;    -   Extensive submacular scar tissue;    -   Significant retinal disease other than subfoveal CNV AMD, such        as diabetic retinopathy or retinal vascular occlusion;    -   Significant non-retinal disease such as ocular atrophy or        cataracts;    -   Known allergy to fluorescein;    -   Current use of prednisolone, other anti-inflammatory steroids or        immune suppression drugs. Non-steroidal drugs such as aspirin        are allowed;    -   Any other significant disease or disorder which, in the opinion        of the Investigator, may either put the participants at risk        because of participation in the study, or may influence the        result of the study, or the participant's ability to participate        in the study;    -   Participants who have participated in another research study        involving an investigational product in the past 12 weeks; and    -   Penicillin sensitivity.

Administration procedure: The pharmaceutical composition containingrAAV.sFlt-1 was administered to study subjects in a setting appropriatefor subretinal injection according to the following procedure:

1. The subject's periocular skin and eyelid margins and eye lashes werecleaned with 5% povidone iodine prior to draping;2. A sterile whole body drape was placed followed by an additional eyedrape.3. Inserted eyelid speculum, ensuring that it is well positionedunderneath the eyelids to direct the eyelashes away from the field andprotected by eye drape.4. Inserted 3×23G or 25G vitrectomy ports;5. Connected saline infusion to 1st port;6. Inserted fiber optic into 2nd port;7. A 36G-41G subretinal cannula was connected to drug syringe viamicroconnector in the 3rd port;8. Under microscopic control, 100 microlitres is injected under theretina;9. Following injection, instruments and ports were withdrawn;10. Chloramphenicol ointment was applied;11. Atropine 1% drop from sterile single use container was instilled;and12. An eye pad and eye shield were applied.

The results of the rAAV.sFlt-1 study are summarized herein.

The eight enrolled subjects (mean age 77 years) all had active subfovealchoroidal neovascularization, with visual acuity of 20/40 to 20/400, andhad previously received between 1 and 25 intravitreal injections ofranibizumab. The patients were randomly distributed into three groups, acontrol group and two experimental groups. All patients receivedintravitreal injections of ranibizumab on day 1 and day 30 of the study.On day 7, 1×10¹⁰ vector genomes of rAAV.sFlt-1 in 100 ul volume wasadministered via subretinal injection to the first experimental groupand 1×10¹¹ vector genomes of rAAV.sFlt-1 in 100 ul volume wasadministered via subretinal injection to the second experimental group.In all six cases for patients in the experimental groups, the bleb ofsub-retinal fluid resolved within 4 hours. After 24 hours, most of theair in the vitreous had absorbed and only the retinal injection siteremained visible. One patient developed a minor hemorrhage associatedwith the procedure that did not affect vision. As expected followingvitrectomy, there was a transient increase in neutrophil counts thatreturned to normal by 14 days post injection. Vector sequence was foundin the tears of one subject at one day post injection that cleared byday 30. Other than this single occurrence, AAV2 was not detected in anyof the subjects' blood, saliva or urine samples either by qPCR or ELISAto date. Background levels of the naturally occurring sFLT-1 proteinshowed a high baseline variation in the urine, serum, and saliva with noincrease following treatment. sFLT-1 levels in the vitreous also variedamong subjects (975-2085 pg/ml). Blood biochemistry, complete bloodcount, and T-cell response, remained without any significant changecompared to baseline. Subretinal injection of rAAV.sFlt-1 showed noclinically significant retinal toxicity as assessed by serial ophthalmicexaminations over a two month period. No superficial, anterior segmentor vitreous inflammatory signs were present in any of the subjects.There was no evidence of visual acuity loss, IOP elevation, retinaldetachment, or any intraocular or systemic immune response in any of thepatients. A summary of anti-VEGF treatments, both initial and rescue,are summarized for each patient in Table 6.

TABLE 6 Summary of Ranibizumab Injections by Patient Day Day Day Day DayDay Day Day Day Day Day Day Day Day Subject 0 30 60 90 120 150 180 210224 252 280 308 336 364 R1001 X X 0 0 0 0 0 No No 0 0 0 0 0 visit visitR1002 X X 0 0 0 0 0 0 0 No 0 0 0 0 visit R1003 X X 0 X X 0 0 X 0 X 0 0 X0 (control) R1004 X X 0 0 0 0 0 0 0 0 0 0 0 X R1005 X X 0 0 0 0 0 No 0 00 0 0 0 visit R1006 X X 0 X No 0 0 0 0 0 0 0 0 0 visit R1007 X X 0 0 0 X0 0 0 0 0 0 0 0 (control) R1008 X X 0 X 0 0 No 0 0 0 0 0 0 0 visit Note:Per protocol, injections at Day 0 and Day 30 were mandatory for allpatients in the study\\

Notably, none of the patients in the experimental groups required rescuetreatment at day 60 and most of the patients in the lower doseexperimental group required 0 rescue treatments at day 90, day 120, day150, day 180 or day 210 or day 270 or day 365 (1 year). The controlpatient required multiple rescue treatments. These results areunexpected and extend the promise of gene therapy for the large cohortof elderly patients suffering from wet AMD. Generally, patients treatedwith current anti-VEGF therapy, such as intravitreal injections of aVEGF inhibitor protein or other anti-VEGF agent will require additionalinjections in 30, 60 or 90 days.

Maximum expression levels of sFLT-1 in a study subject or a patient arereached six to eight weeks after subretinal administration ofrAAV.sFLT-1. During this so called “ramp-up” period, at least one, twoor three intravitreal injections of an anti-VEGF agent are injected at15 to 45 day intervals, and preferably about 30 day intervals, toprevent disease progression. It is preferred to administer the firstintravitreal injection of an anti-VEGF agent between 1 to 30 days, andpreferably between 5 to 10 days, prior to administration of rAAV.sFlt-1to allow for absorption of the intravitreally injected anti-VEGF agent(Lucentis or Avastin or Eylea or other non sFLT agents). If this firstintravitreal injection is administered less than 24 hours prior tosubretinal administration of rAAV.sFLT, it may be washed out of thevitreous during the subretinal injection procedure leading to asub-therapeutic anti-VEGF agent concentration and disease progression.

After the completion of the ramp period, patients who express sufficientsFLT-1 to treat or prevent progression of their AMD may not needadditional intravitreal anti-VEGF injections although it is expect thatthey will remain under the care of a physician. Patients are monitoredand treated on an as-needed basis based on objective criteria, such asan increased center point retinal thickness measurement with an opticalcoherence tomography.

In this study, patients in the control and both experimental groups wereevaluated for signs of active choroidal neovascularization on anapproximately monthly basis and retreated with intravitreal ranibizumabif any of the following criteria was met:

-   -   >10 Early Treatment Diabetic Retinopathy Study (ETDRS) letter        loss from subject's previous visit (attributable to retinal        causes), OR a decrease of >5 ETDRS letters from previous visit        in conjunction with patient perception of functional loss;    -   Any increased, new, or persistent subsensory, sub-Retinal        Pigment Epithelial (RPE), or intraretinal fluid on OCT;    -   Signs of increased CNV leakage via FA.

Example 13 Optical Coherence Tomography (OCT)

Spectral Domain Optical Coherence Tomography (SD-OCT) was performedusing approved equipment (Heidelberg Spectralis® SD-OCT) and standardtechniques to monitor center point retinal thickness and fluid leakagein the retina of patients.

Optical Coherence Tomography (OCT) is a non-contact medical imagingtechnology similar to ultrasound and MRL With OCT, reflected light isused to produce detailed cross-sectional and 3D images of the eye. TheSPECTRALIS® SD-OCT simultaneously measures multiple wavelengths ofreflected light across a spectrum, hence the name spectral domain. Theincreased speed and number of scans translates into higher resolutionand a better chance of observing disease. In patients with wet AMD, thedetection of new retinal fluid or a clinically significant increase inretinal thickness may be detected by SD-OCT. (Adhi et al., Curr OpinOphthalmol. 2013 May; 24(3):213-21; Malamos et al., Invest Ophthalmol Vis Sci. 2009 October; 50(10):4926-33). Detection of these symptoms in apatient with AMD indicates disease progression that warrants treatmentwith an anti-VEGF therapy such as Lucentis or Eylea.

The retinal health and symptoms of AMD progression of each subject inthe study were monitored via SD-OCT. At least 6 radial scans through themacula, each approximately 6 mm in length, were taken; and OCTimages/scans were collected at each specified visit. The SD-OCT imageswere evaluated for the presence of intraretinal fluid by a masked readerand the central retinal thickness was measured using Heidelberg HeyexSD-OCT software. The central retinal thickness results for each visitfor 8 patients are presented below in Table 7.

TABLE 7 Mean Change in Central Retinal Thickness from Baseline at Day 0in microns by dosing group Study Day 14 28 56 84 112 140 168 196 224 252280 308 336 364 Control −101 24 −194 −178 −176 −199 −131 −124 −186 −190−198 −172 −157 −138 Low dose −140 −115 −161 −189 −173 −163 −157 −147−149 −155 −161 −144 −127 −134 High Dose −254 −245 −266 −254 −245 −239−235 −209 −219 −225 −215 −239 −246 −245As shown in table 7, the mean central retinal thickness of the subjectsin all dosing cohorts decreased after administration of the intravitrealinjections of the anti-VEGF protein (Lucentis) at the beginning of thestudy as required by protocol. As expected, the central retinalthickness of the patients in the control group starts to increase andfluid can be seen on SD-OCT images within 30-90 days of theadministration of the anti-VEGF protein. Unexpectedly, the centralretinal thickness of the subjects in the low and high dosing groups isgenerally well controlled by rAAV.sFlt-1 and does not increase overtime. New intraretinal fluid does not occur in the retinas of the lowdose group subjects or the high dose group subjects. This is shown byOCT, for example, in FIG. 24. At 12 months, the central retinalthickness of subjects treated with rAAV.sFlt-1 did not increase by morethan 50 microns, or by more than 100 microns, or by more than 250microns within 12 months of administration of a pharmaceuticalcomposition comprising rAAV.sFlt-1. When compared against baseline, thecentral retinal thickness of human subjects treated with rAAV.sFlt-1decreased by 50 microns or in some cases by 100 microns or in somecases, by 200 microns. This decrease was observed within 8 weeks ofadministering sFlt-1 and was maintained at 3 months, 6 months, 9 monthsand 12 months. This result is surprising and is unknown in in theclinical treatment of AMD and ocular neovascularization in humansubjects. More generally, without additional administrations of ananti-VEGF protein or other VEGF inhibitor, intraretinal fluid and anincrease in central retinal thickness will be observed with 30 days, 60days, 90 days or 180 days of an initial anti-VEGF treatment.

Fluorescein Angiography (FA)

FA was performed using a standard technique. Transit images are taken ofthe study eye. Mid and late phase images are taken of the study andnon-study eye; and FA is be obtained at each specified visit.

Biodistribution Studies

Dissemination of vector was investigated by polymerase chain reaction(PCR) amplification of vector genomes isolated from samples of tears,plasma, urine and saliva. Biodistribution of vector and sFLT-1 wasinvestigated by ELISA for sFLT-1 and AAV2 capsids in plasma, tears andsaliva.

Extraction of DNA

Samples (100-300 ul) were pipetted onto Sample Collection Cards (Qiagen,Valencia, Calif.) or sterile foam tip applicators. DNA was extractedfrom each sample as per manufacturer's protocol. Purified DNA wasdissolved in 50 ul of elution buffer. The amount of DNA present wasdetermined by spectrophotometry.

Detection of rAAV.sFlt-1 by Real Time PCR

Genomic DNA samples (0.5-1 μg) were screened for the presence of theAAV.sFlt-1 vector using the TaqMan® Gene Expression Assays (AppliedBiosystems, U.S.A.). The assay consists of a pair of unlabeled PCRprimers which amplifies a fragment between the AAV2 and the sFLT-1sequences, and a TaqMan® probe with a FAM™ or VIC® dye label and minorgroove binder moiety on the 5′ end, and non-fluorescent quencher dye onthe 3′ end. The cycling conditions were 1 hold for 2 minutes at 50° C.and another hold at 95° C. for 20 seconds, followed by 45 cycles of 95°C. for 3 seconds and 60° C. for 30 seconds.

Samples positive for the rAAV.sFlt-1 fragment were further tested andthe gene copy number of rAAV.sFlt-1 present were quantified by real timepolymerase chain reaction (PCR). Between 0.5-1.0 ug of extracted DNAwere amplified in 20-ul reaction mixes containing Platinum SYBR GreenqPCR Supermix-UDG (Invitrogen, Carlsbad, Calif., USA) and 0.5 uM of eachprimer using the IQ5 Bio-Rad real-time PCR system (Bio-Rad, Hercules,Calif., USA). A similar set of samples spiked with plasmid DNAcontaining the target sequence was set up in parallel as the spikedsamples. The primer pair used (forward: CACTAGTCCAGTGTGGTGGA (SEQ IDNO:123); reverse: AGCCAGGAGACAACCACTTC (SEQ ID NO:124)) was designedwith the aid of Primer3 Output (Whitehead Institute, MA, USA) to amplifythe region from the vector cDNA into the sFLT-1 gene using the Rotorgene(Corbett). The cycling conditions that were used were: 2 min 50.0° C., 2min 95.0° C. and 60 three-step cycles of 95.0° C. 20 s, 60.0° C. for 20s and 72.0° C. for 20 s. A standard curve was generated in each run from10-fold dilutions of plasmid DNA (pSSV.sFlt-1) which had the same targetvector sequence. Each sample was analyzed in triplicate.

Quantifying sFlt-1 Protein Concentration by ELISA

The concentration of sFLT-1 present in the plasma, tears and saliva weremeasured quantitatively by ELISA using a Quantikine ELISA kit (R&DSystems, Minneapolis, Minn.) which was based on the sandwich immunoassaytechnique. The samples (100 ul) were added to the 96-well plate coatedwith a monoclonal antibody specific for VEGF R1/sFLT-1 and allowed toincubate for 2 hours. Any unbound sFLT-1 was removed by washing with abuffer. Following incubation with an enzyme-linked polyclonal antibodyspecific for VEGF R1/sFLT-1, the excess of antibody-enzyme conjugate waswashed off and the samples were then be incubated with a substratesolution. Enzyme-catalyzed chromogen formation was quantified bymeasuring the visible absorbance at 450 nm. The concentrations of sFLT-1(in pg/ml) in the samples were calculated from the absorbance valueusing a calibration curve plotted with recombinant human sFLT-1.

Detection of AAV2 by ELISA

Presence of AAV2 capsid in the plasma, tears, urine and saliva wasanalyzed using the AAV2 Titration ELISA Kit (American Research Products,Inc., Belmont, Mass., USA). This kit is based on a sandwich ELISAtechnique and uses a mouse monoclonal antibody specific for aconformational epitope on assembled AAV particles. This monoclonalantibody is coated onto microplate strips and is used to capture AAVparticles from the specimen. Captured AAV particles were detected in twosteps. First a biotin-conjugated monoclonal antibody to AAV was bound tothe immune complex. In the second step streptavidin peroxidase conjugatereacts with the biotin molecules. Addition of substrate solution resultsin a color reaction which was proportional to specifically bound virusparticles. The absorbance was measured photometrically at 450 nm. Thekit control provided contains an AAV particle preparation of emptycapsids and it allowed the quantitative determination of samples of anunknown particle titer. Samples (100 ul) were added to the plates andthe assay was to be carried out according to the manufacturer'sprotocol.

Detection of Neutralizing AAV-2 Antibody

Plasma was assayed for the ability to block the transduction of HEK293cells with AAV2.gfp. Patient's plasma was serially diluted in normalmouse serum in multi-well plates. AAV2.gfp was added to each well andplates were incubated at 37° C. for 1 hour before addition to HEK293cells in triplicate. The neutralizing antibody titer was expressed asthe plasma dilution that resulted in 50% inhibition of transduction byAAV2-gfp. Maximum gfp activity was represented by vector diluted innormal mouse serum; maximum inhibition was represented by medium only innormal mouse serum. Baseline plasma from each subject was assayedalongside each post-op sample. Green cells from transduction of 293Tcells with AAV2.gfp were counted in the test wells after 48 hours andcompared with the number of green cells in the baseline serum sample.

Detection of Anti-AAV2 Antibodies

To detect plasma antibodies to AAV2 capsid, enhanced protein-bindingELISA plates were coated with 10⁹ vg/ml of AAV2 (Vector Core Facility,North Carolina) at 4° C. overnight. The plates were be blocked at 37° C.for 2 hours and then are incubated at 4° C. overnight with seriallydiluted anti-AAV2 monoclonal antibody (Industries International,Concord, Mass.) or 1:50, 1:100, 1:200, or 1:400 dilutions of patientplasma. The plates were incubated with horse radish peroxidase(HRP)-conjugated anti-human Ig at 37° C. for 2 hours, then withtetramethyl benzidine (TMP) substrate and hydrogen peroxide (H2O2). Thereaction was stopped by phosphoric acid (H3PO4) and read at 450 nm on aplate reader. The titer of anti-AAV2 antibodies were calculated based onthe standard curve of the commercial antibody determined in parallel.Each value was determined in triplicate.

Geographic Atrophy

The human study subjects were examined for signs of geographic atrophyin their treated and untreated eyes according to standard techniques.Increases geographic atrophy was not observed in patients treated withrAAV.sFlt-1 at 3 months, 6 months, 9 months, or 12 months. It ishypothesized that the treatment may stop progression of geographicatrophy in a treated eye for up to 15 months, 18 months, 24 months, 36months, 5 years and 10 years.

Example 14

To further test the safety and efficacy of rAAV.sFlt-1 for the treatmentof wet AMD and choroidal neovascularization, forty (40) additionalsubjects were enrolled in a controlled clinical study. As in Example 12,rAAV.sFlt-1 was produced in accordance with FDA and ICH guidelines atthe UNC Vector Core Human Application Laboratory. Eligibility, inclusionand exclusion criteria for the study were as follows:

Eligibility:

-   -   Ages Eligible for Study: 55 Years and older    -   Genders Eligible for Study: Both    -   Accepts Healthy Volunteers: No

Inclusion Criteria:

-   -   Age greater than or equal to 55 years;    -   Subfoveal CNV secondary to AMD and with best corrected visual        acuity in the study eye of 20/30-20/400 and 20/200 or better in        the other eye;    -   Fluorescein angiogram of the study eye must show evidence of a        leaking subfoveal choroidal neovascular lesion; or choroidal        neovascularization currently under active management with        anti-VEGF therapy;    -   Must be a candidate for anti-VEGF intravitreal injections;    -   The entire dimension of the lesion must not exceed 12 Macular        Photocoagulation Study disc areas;    -   No previous retinal treatment of photodynamic therapy or laser;    -   Able to provide informed consent;    -   Participant has clinically acceptable laboratory parameters and        ECG at the time of enrollment; and    -   Able to comply with protocol requirements, including follow-up        visits.

Exclusion Criteria:

-   -   Liver enzymes >2× upper limit of normal;    -   Clinical evidence of active infection of any type, including        adenovirus, hepatitis A, B, or C, or HIV virus; or documented        history of hepatitis B or hepatitis C;    -   Any prior treatment for AMD in the study/control eye, excluding        anti-VEGF injections;    -   A tear in the retinal pigmented epithelium;    -   Extensive sub-fovial scarring, extensive geographic atrophy, or        thick subretinal blood in the study eye as determined by the        investigator;    -   Significant retinal disease other than subfoveal CNV AMD, such        as diabetic retinopathy or retinal vascular occlusion, that        could compromise vision in the study eye;    -   Significant non-retinal disease such as ocular atrophy or        significant cataract in the study eye, including central corneal        scarring that affects visual acuity, glaucoma with field        defects, or any measurable uveitis;    -   Known allergy to fluorescein;    -   Current use of prednisolone, other anti-inflammatory steroids or        immune suppression drugs. Inhaled steroids and non-steroidal        drugs such as aspirin are allowed;    -   Any other significant disease or disorder which, in the opinion        of the Investigator, may either put the participants at risk        because of participation in the study, or may influence the        result of the study, or the participant's ability to participate        in the study;    -   Participants who have participated in another research study        involving an investigational product in the past 12 weeks; and    -   Penicillin sensitivity confirmed by participant medical records.

Initial enrolled subjects had active subfoveal choroidalneovascularization, with visual acuity in the study eye of 20/30 to20/400, and had previously received between 0 and 25 intravitrealinjections of ranibizumab. The patients were randomly distributed into acontrol group or an experimental group until a total of 14 patientscontrol patients and 26 experiments patients were enrolled. All patientsreceived intravitreal injections of ranibizumab on day 1 and day 30 ofthe study. On day 7, 1×10¹¹ vector genomes of rAAV.sFlt-1 in 100 ulvolume was administered via subretinal injection to the experimentalgroup.

As in the study in Example 12, maximum expression levels of sFLT-1 in astudy subject or a patient were reached six to eight weeks aftersubretinal administration of rAAV.sFLT-1. During this so called“ramp-up” period, at least one, two or three intravitreal injections ofan anti-VEGF agent were injected at 15 to 45 day intervals, andpreferably about 30 day intervals, to prevent disease progression. It ispreferred to administer the first intravitreal injection of an anti-VEGFagent between 1 to 30 days, and preferably between 5 to 10 days, priorto administration of rAAV.sFLT-1 to allow for absorption of theintravitreally injected anti-VEGF agent (Lucentis or Avastin or Eylea orother non sFLT agents). If this first intravitreal injection isadministered less than 24 hours prior to subretinal administration ofrAAV.sFLT, it may be washed out of the vitreous during the subretinalinjection procedure leading to a sub-therapeutic anti-VEGF agentconcentration and disease progression.

After the completion of the ramp period, patients who expressedsufficient sFLT-1 to treat or prevent progression of their AMD or othersymptoms of choroidal neovascularization did not need additionalintravitreal anti-VEGF injections although it is expected that they willremain under the care of a physician.

In this study recited in this example, patients in the control andexperimental groups were evaluated for signs of active choroidalneovascularization on an approximately monthly basis and retreated withintravitreal ranibizumab if any of the following criteria was met:

-   -   >10 Early Treatment Diabetic Retinopathy Study (ETDRS) letter        loss from subject's previous visit (attributable to retinal        causes), OR a decrease of >5 ETDRS letters from previous visit        in conjunction with patient perception of functional loss;    -   Any increased, new, or persistent subsensory, sub-Retinal        Pigment Epithelial (RPE), or intraretinal fluid on OCT;    -   Signs of increased CNV leakage via FA.

Example 15

To test the safety and efficacy of rAAV.sFlt-1 for the prevention orprophylaxis of the ocular neovascular disease Age Related Maculardegeneration (AMD), an additional controlled clinical study with forty(150) patients is conducted. rAAV(bv).sFlt-1 is produced in accordancewith FDA and ICH guidelines at Lonza Houston, Inc. (Houston, Tex.).Eligibility, inclusion and exclusion criteria for the study were asfollows:

Eligibility:

-   -   Ages Eligible for Study: 50 Years and older    -   Genders Eligible for Study: Both    -   Accepts Healthy Volunteers: Yes

Inclusion Criteria:

-   -   Patients with nonexudative AMD (either categories 2, 3 or 4        according to the AREDS criteria; in group 4 the eyes with        no-advanced AMD will be included); Patients with AMD classified        as either “wet” or “dry” are included;    -   Age between 50 and 90 years;    -   Able to understand and comply with the requirements of the        trial;    -   Visual acuity >0.4;

Exclusion Criteria:

-   -   Currently enrolled in an ophthalmic clinical trial;    -   Eyes with concomitant macular or choroidal disorders other than        AMD and with indefinite signs of AMD;    -   Eyes with a diagnosis of exudative AMD with active subretinal        neovascularization (SRNV) or CNV lesions requiring laser        photocoagulation in the study eye;    -   Subjects with significant ocular lens opacities causing vision        decrease;    -   Subjects with amblyopia;    -   Subjects with optic nerve disease (neuropathy, atrophy,        papilledema), unstable glaucoma as defined by intraocular        pressures greater than 25 mm Hg, 3 or more glaucoma medications,        C/D of 0.8 or greater and visual fields consistent with        glaucoma; history of retina-vitreous surgery, degenerative        myopia, active posterior intraocular inflammatory disease,        chronic use of topical ocular steroid medications,        vasoproliferative retinopathies (other than AMD), rhegmatogenous        retinal detachment, and inherited macular dystrophies;    -   Subjects with demand type pacemakers or epilepsy;    -   Subjects with uncontrolled hypertension (defined as diastolic of        90 or greater and systolic of 150 or greater);    -   Subjects with recent history (within the previous year) of        cerebral vascular disease;    -   manifested with transient ischemic attacks (TIA's) or cerebral        vascular accidents (CVA's);    -   Subjects with a history of AIDS;    -   Subjects who have had intraocular surgery in trial eye within 3        months prior to enrolling in the trial;    -   Patients who are unwilling to adhere to visit examination        schedules;

Primary Outcome Measures:

-   -   MPOD and multifocal electroretinograms [Time Frame: 1        year][Designated as safety issue: Yes]

Secondary Outcome Measures:

-   -   The safety and efficacy of rAAV(bv).sFlt-1 in reducing the risk        of the development of advanced AMD. [Time Frame: 1        year][Designated as safety issue: Yes]

TABLE 9 Experimental Design Arms Arms Assigned Interventions ActiveComparator: Group I Drug: of rAAV(bv).sFlt-1 1 × 10¹⁰ vector genomes ofrAAV(bv).sFlt-1 in 100 ul 1 × 10¹⁰ vector genomes of rAAV.sFlt-1 involume is administered via subretinal injection to the 100 ul volume isadministered via subretinal experimental group within 30-90 dayintervals for 36 injection to the experimental group. months ActiveComparator: Group II Drug: of rAAV(bv).sFlt-1 1 × 10¹¹ vector genomes ofrAAV(bv).sFlt-1 in 100 ul 1 × 10¹¹ vector genomes of rAAV.sFlt-1 involume is administered via subretinal injection to the 100 ul volume isadministered via subretinal experimental group within 180-365 dayintervals for 36 injection to the experimental group. months PlaceboComparator: Group Placebo Drug Placebo: Saline solution Drug Placebo:Saline solution Drug Placebo: Saline solution, until one year. Patientson placebo showing early stages of AMD may receive rAAV(bv).sFlt-1Active Comparator: Ranibizumab 0.3 mg Drug: Ranibizumab Patients receiveranibizumab 0.3 mg monthly Sterile solution for intravitreal injection.administered intravitreally for 36 months. Other Name: Lucentis

Example 16

To test the safety and efficacy of rAAV.sFlt-1 for the treatment of theocular neovascular disease Diabetic Macular Edema (DME), an additionalcontrolled clinical study with forty (40) patients is conducted.rAAV(bv).sFlt-1 is produced in accordance with FDA and ICH guidelines atLonza Houston, Inc. (Houston, Tex.). Eligibility, inclusion andexclusion criteria for the study were as follows:

Eligibility:

-   -   Ages Eligible for Study: 18 Years and older    -   Genders Eligible for Study: Both    -   Accepts Healthy Volunteers: No

General Inclusion Criteria:

-   -   Subjects are eligible if the following criteria are met:    -   Willingness to provide written informed consent and, at U.S.        sites, Health Insurance Portability and

Accountability Act (HIPAA) authorization, and in other countries, asapplicable according to national laws.

-   -   Diabetes mellitus (Type 1 or 2).    -   Retinal thickening secondary to diabetes mellitus (DME)        involving the center of the fovea with central macular thickness        ≧275 μm in the center subfield as assessed on optical coherence        tomography (OCT).    -   Best corrected visual acuity (BCVA) score in the study eye of        20/40 to 20/320 approximate Snellen equivalent using the Early        Treatment Diabetic Retinopathy Study (ETDRS) protocol at an        initial testing distance of 4 meters.    -   Decrease in vision determined to be primarily the result of DME        and not to other causes.    -   Ability (in the opinion of the investigator) and willingness to        return for all scheduled visits and assessments.

Exclusion Criteria:

-   -   History of vitreoretinal surgery in the study eye.    -   Panretinal photocoagulation (PRP) or macular laser        photocoagulation in the study eye within 3 months of screening.    -   Proliferative diabetic retinopathy (PDR) in the study eye, with        the exception of inactive, regressed PDR.    -   Iris neovascularization, vitreous hemorrhage, traction retinal        detachment, or preretinal fibrosis involving the macula in the        study eye.    -   Vitreomacular traction or epiretinal membrane in the study eye.    -   Ocular inflammation (including trace or above) in the study eye.    -   History of idiopathic or autoimmune uveitis in either eye.    -   Structural damage to the center of the macula in the study eye        that is likely to preclude improvement in VA following the        resolution of macular edema, including atrophy of the retinal        pigment epithelium (RPE), subretinal fibrosis, or organized        hard-exudate plaque.    -   Ocular disorders in the study eye that may confound        interpretation of study results, including retinal vascular        occlusion, retinal detachment, macular hole, or choroidal        neovascularization (CNV) of any cause (eg, age-related macular        degeneration (AMD), ocular histoplasmosis, or pathologic        myopia).    -   Cataract surgery in the study eye within 3 months,        yttrium-aluminum-garnet (YAG) laser capsulotomy within the past        2 months, or any other intraocular surgery within the 90 days        preceding Day 0.    -   Uncontrolled glaucoma or previous filtration surgery in the        study eye.    -   Uncontrolled blood pressure.    -   History of cerebral vascular accident or myocardial infarction        within 3 months prior to Day 0.    -   Uncontrolled diabetes mellitus.    -   Renal failure requiring dialysis or renal transplant.    -   History of other disease, metabolic dysfunction, physical        examination finding, or clinical laboratory finding giving        reasonable suspicion of a disease or condition that        contraindicates the use an investigational drug, might affect        interpretation of the results of the study, or renders the        subject at high risk from treatment complications.

Primary Outcome Measures:

-   -   Percentage of Patients Who Gain ≧15 Letters in Their Best        Corrected Visual Acuity (BCVA) Score

From Baseline at Month 12 [Time Frame: Baseline to Month 12][Designatedas safety issue: No]

Secondary Outcome Measures:

-   -   Mean Change From Baseline in Best Corrected Visual Acuity (BCVA)        Score at Months 12, 24 and 36    -   Percentage of Patients With a Visual Acuity (VA) Snellen        Equivalent of 20/40 or Better at Months 12, 24 and 36.    -   Mean Change From Baseline in Central Foveal Thickness as        measured by SD-OCT at Months 12, 24 and 36.    -   Reduction in Frequency of concomitant anti-VEGF treatment ((e.g.        Lucentis, Avastin, Macugen or Eyelea) [Designated as safety        issue: No]

TABLE 10 Experimental Design Arms for DME Study Arms AssignedInterventions Experimental: I Low Dose Drug: rAAV.sFlt-1 (AVA-01) 1 ×10¹⁰ vector genomes of rAAV(bv).sFlt-1 in 100 ul volume 1 × 10¹⁰ vectorgenomes of rAAV.sFlt-1 was administered via subretinal injection to theexperimental in 100 ul volume was administered via group. subretinalinjection to the experimental Follow-up phase: Participants onrAAV.sFlt-1 are monitored group. monthly and receive rescue treatmentswith intravitreal anti- VEGF therapy if they meet the study criteria forretreatment. Experimental: II High Dose Drug: rAAV.sFlt-1 (AVA-01) 1 ×10¹¹ vector genomes of rAAV.sFlt-1 in 100 ul volume 1 × 10¹¹ vectorgenomes of rAAV.sFlt-1 was administered via subretinal injection to theexperimental in 100 ul volume was administered via group. subretinalinjection to the experimental Follow-up phase: Participants onrAAV.sFlt-1 are monitored group. monthly and receive rescue treatmentswith intravitreal anti- VEGF therapy if they meet the study criteria forretreatment. Active Comparator: Ranibizumab injection 0.3 mg. Drug:Ranibizumab Participants receive two initial injections of Ranibizumabat Sterile solution for intravitreal injection. Day 0 and Day 30. OtherName: Lucentis Follow-up phase: Participants are monitored monthly andreceive rescue treatments with Ranibizumab if they meet the studycriteria for retreatment.

Initial enrolled subjects have DME, with visual acuity in the study eyeof 20/40 to 20/320, and will have previously received between 0 and 25intravitreal injections of ranibizumab or aflibercept. The patients arerandomly distributed into a control group or two experimental groupsuntil a total of 14 patients control patients and 13 low doseexperimental patients and 13 high dose experimental patients areenrolled. All patients received intravitreal injections of ranibizumabon day 1 and day 30 of the study. On day 7, 1×10¹⁰ or 1×10¹¹ vectorgenomes of rAAV(bv).sFlt-1 in 100 ul volume are administered viasubretinal injection to the experimental groups.

As in the study in Example 12, maximum expression levels of sFLT-1 in astudy subject or a patient are reached are six to eight weeks aftersubretinal administration of rAAV(bv).sFLT-1. During this so called“ramp-up” period, at least one, two or three intravitreal injections ofan anti-VEGF agent are injected at 15 to 45 day intervals, andpreferably about 30 day intervals, to prevent disease progression. It ispreferred to administer the first intravitreal injection of an anti-VEGFagent between 1 to 30 days, and preferably between 5 to 10 days, priorto administration of rAAV(bv).sFLT-1 to allow for absorption of theintravitreally injected anti-VEGF agent (Lucentis or Avastin or Eylea orother non sFLT agents). If this first intravitreal injection isadministered less than 24 hours prior to subretinal administration ofrAAV(bv).sFLT, it may be washed out of the vitreous during thesubretinal injection procedure leading to a sub-therapeutic anti-VEGFagent concentration and disease progression.

After the completion of the ramp period, patients who express sufficientsFLT-1 to treat or prevent progression of their DME may not needadditional intravitreal anti-VEGF injections although it is expect thatthey will remain under the care of a physician.

In this study recited in this example, patients in the control andexperimental groups are evaluated for signs of active or new DME andneovascularization on an approximately monthly basis and are retreatedwith intravitreal ranibizumab if any of the following criteria was met:

-   -   >10 Early Treatment Diabetic Retinopathy Study (ETDRS) letter        loss from subject's previous visit (attributable to retinal        causes), OR a decrease of >5 ETDRS letters from previous visit        in conjunction with patient perception of functional loss;    -   Any increased, new, or persistent subsensory, sub-Retinal        Pigment Epithelial (RPE), or intraretinal fluid on OCT;    -   Signs of increased CNV leakage via FA.

Example 17

To test the safety and efficacy of rAAV.sFlt-1 for the treatment of theocular neovascular disease Retinal Vein Occlusion (RVO), an additionalcontrolled clinical study with forty (40) patients is conducted. Theclinical study is performed with patients of 2 cohorts, 1 cohortincluding patients with Central Retinal Vein Occlusion (CRVO) and 1cohort including Branched Retinal Vein Occlusion (BRVO). As in Example15, rAAV(bv).sFlt-1 is produced in accordance with FDA and ICHguidelines at Lonza Houston, Inc. (Houston, Tex.). Eligibility,inclusion and exclusion criteria for the study were as follows:

Inclusion Criteria:

-   -   Center-involved macular edema secondary to central retinal vein        occlusion (CRVO) or Branch-involved macular edema secondary to        BRVO for no longer than 9 months with mean central subfield        thickness ≧250 μm on optical coherence tomography (OCT);    -   Adults ≧18 years;    -   Early treatment diabetic retinopathy study (ETDRS) best        corrected visual acuity (BCVA) of 20/40 to 20/320 (73 to 24        letters) in the study eye;

Exclusion Criteria:

-   -   Any prior treatment with anti-VEGF agents in the study eye        (Pegaptanib sodium, anecortave acetate, bevacizumab,        ranibizumab, etc.) or previous administration of systemic        anti-angiogenic medications;    -   Prior panretinal laser photocoagulation or macular laser        photocoagulation in the study eye    -   CRVO disease duration >9 months from date of diagnosis; BRVO        disease duration >9 months from date of diagnosis;    -   Previous use of intraocular corticosteroids in the study eye or        use of periocular corticosteroids in the study eye within the 3        months prior to Day 1;    -   Iris neovascularization, vitreous hemorrhage, traction retinal        detachment, or preretinal fibrosis involving the macula in        either the study eye or fellow eye;

Primary Outcome Measures:

-   -   Mean Change From Baseline in Best Corrected Visual Acuity (BCVA)        Score at 6 Months. [Time Frame: Baseline and 6        months][Designated as safety issue: No].    -   Defined study baseline range of Early Treatment Diabetic        Retinopathy Study (ETDRS) Best Corrected Visual Acuity (BCVA)        letter score of 73 to 24 (=Acuity of 20/40 to 20/320) in the        study eye; a higher score represents better functioning.        Nominator=(Number of participants who maintained vision*100);        Denominator=Number of participants analyzed.

Secondary Outcome Measures:

-   -   Percentage of Participants Who Gained ≧15 Letters in BCVA Score        at Month 6 Compared With Baseline.    -   Mean Change From Baseline in Central Retinal Thickness (CRT) at        6 months [Time Frame: Baseline and 6 months][Designated as        safety issue: No]    -   Reduction in frequency of concomitant anti-VEGF treatment ((e.g.        Lucentis, Avastin, Macugen or Eyelea) [Designated as safety        issue: No]

TABLE 11 Experimental Design Arms for BRVO/CRVO Study Arms AssignedInterventions Experimental: 1 × 10¹⁰ vector genomes of rAAV.sFlt-1Biological: 1 × 10¹⁰ vector genomes of in 100 ul volume is administeredvia subretinal injection to rAAV.sFlt-1. Subretinal injection. theexperimental group, on Day 7. Drug: Ranibizumab injection 0.3 mg ifFollow-up phase: Participants on rAAV.sFlt-1 are monitored meetreinjection criteria. monthly and receive rescue treatments withintravitreal anti- VEGF therapy if they meet the study criteria forretreatment. Experimental: 1 × 10¹¹ vector genomes of rAAV.sFlt-1Biological: 1 × 10¹¹ vector genomes of in 100 ul volume is administeredvia subretinal injection to rAAV.sFlt-1. Subretinal injection. theexperimental group, on Day 7. Drug: Ranibizumab injection 0.3 mg ifFollow-up phase: Participants on rAAV.sFlt-1 are monitored meetreinjection criteria. monthly and receive rescue treatments withintravitreal anti- VEGF therapy if they meet the study criteria forretreatment. Active Comparator: Ranibizumab injection 0.3 mg. Drug:Ranibizumab injection 0.3 mg Participants receive two initial injectionsof Ranibizumab at Ranibizumab injection 0.3 mg in a single- Day 0 andDay 30. dose regimen given at Day 0 and Day 30. Follow-up phase:Participants are monitored monthly and Other Name: Lucentis receiverescue treatments with Ranibizumab if they meet the study criteria forretreatment.

Initial enrolled subjects have CRVO or BRVO, with visual acuity in thestudy eye of 20/40 to 20/320, and will have previously received between0 and 25 intravitreal injections of ranibizumab or aflibercept. Thepatients are randomly distributed into a control group or twoexperimental groups until a total of 14 patients control patients and 13low dose experimental patients and 13 high dose experimental patientsare enrolled. All patients received intravitreal injections ofranibizumab on day 1 and day 30 of the study. On day 7, 1×10¹⁰ or 1×10¹¹vector genomes of rAAV(bv).sFlt-1 in 100 ul volume are administered viasubretinal injection to the experimental groups.

As in the study in Example 14, maximum expression levels of sFLT-1 in astudy subject or a patient are reached are six to eight weeks aftersubretinal administration of rAAV(bv).sFLT-1. After the completion ofthe ramp period, as described in Example 14, patients who expresssufficient sFLT-1 to treat or prevent progression of their BRVO or CRVOmay not need additional intravitreal anti-VEGF injections although it isexpect that they will remain under the care of a physician.

In this study recited in this example, patients in the control andexperimental groups are evaluated for signs of active or new retinalvein occlusion and neovascularization on an approximately monthly basisand are retreated with intravitreal ranibizumab if any of the followingcriteria was met:

-   -   >10 Early Treatment Diabetic Retinopathy Study (ETDRS) letter        loss from subject's previous visit (attributable to retinal        causes), OR a decrease of >5 ETDRS letters from previous visit        in conjunction with patient perception of functional loss;    -   Any increased, new, or persistent subsensory, sub-Retinal        Pigment Epithelial (RPE), or intraretinal fluid on OCT;    -   Signs of increased CNV leakage via FA.

1-41. (canceled)
 42. A method for the treatment or prophylaxis of ocularneovascularization in a human subject having or at risk for developingocular neovascularization, the method comprising: administering to theretina of the human subject an effective amount of a pharmaceuticalcomposition comprising a recombinant virus.
 43. The method according toclaim 42, wherein the recombinant virus comprises a nucleic acidsequence encoding an anti-VEGF protein.
 44. The method according toclaim 43, wherein the anti-VEGF protein comprises a functional fragmentof human sFLT1 having a sequence identity of 90% or more to SEQ IDNO:109.
 45. The method according to claim 42, wherein the recombinantvirus is AAV.
 46. The method according to claim 42, wherein theadministering of the pharmaceutical composition is performed at afrequency less than 3 times a year in the human subject.
 47. The methodaccording to claim 42, wherein the administering of the pharmaceuticalcomposition is performed only once in at least 18 months.
 48. The methodaccording to claim 42, wherein said administering is at a dose of atmost 1×10¹¹ vector genomes.
 49. The method according to claim 42,wherein said administering is at a dose of at least 1×10⁸ vectorgenomes.
 50. The method of claim 42, wherein a reduction inneovascularization is observed by Fluorescein Angiography (FA) followingthe administering of the pharmaceutical composition.
 51. The method ofclaim 49, further comprising performing FA after the administering ofthe pharmaceutical composition, wherein a reduction inneovascularization is observed by FA.
 52. The method of claim 42,wherein the best corrected visual acuity (BCVA) of the human subjectdecreases by fewer than 15 letters as measured by Early TreatmentDiabetic Retinopathy Study (ETDRS) letters following the administeringof the pharmaceutical composition.
 53. The method of claim 52, furthercomprising measuring visual acuity by ETDRS letters after theadministering of the pharmaceutical composition, wherein the BCVA of thehuman subject decreases by fewer than 15 ETDRS letters.
 54. The methodof claim 42, wherein the best corrected visual acuity (BCVA) of thehuman subject improves by at least 1 line as measured by Early TreatmentDiabetic Retinopathy Study (ETDRS) letters following the administeringof the pharmaceutical composition.
 55. The method of claim 54, furthercomprising measuring visual acuity by ETDRS letters after theadministering of the pharmaceutical composition, wherein the BCVA of thehuman subject improves by at least 1 line as measured by ETDRS letters.56. The method according to claim 42, wherein the administeringcomprises subretinal injection.
 57. The method according to claim 56,wherein the subretinal injection is within the central retina.
 58. Themethod according to claim 56, further comprising removal of the vitreousgel prior to administering the pharmaceutical composition.
 59. Themethod according to claim 42, wherein said administering comprisesinjecting the pharmaceutical composition over a period of less than 1minute.
 60. The method according to claim 42, wherein said administeringcomprises injecting the pharmaceutical composition over a period of 1-3minutes.
 61. The method according to claim 42, wherein saidadministering comprises injecting the pharmaceutical composition at arate of about 35 μl/min to about 65 μl/min.
 62. The method according toclaim 42, wherein said administering comprises injecting thepharmaceutical composition at a rate of about 65 μl/min to about 150μl/min.
 63. The method according to claim 42, wherein the method is forthe treatment of ocular neovascularization and the human subject hasocular neovascularization.
 64. The method according to claim 42, whereinthe method is for the prevention of ocular neovascularization and thehuman subject is at risk for developing ocular neovascularization.